Ganglioside derivatives

ABSTRACT

New functional ganglioside derivatives comprising ester, amide and peracylated derivatives of gangliosides, preparation procedures therefor, pharmaceutical preparations containing the same, and the therapeutic use of the derivatives.

This application is a continuation of copending application Ser. No.749,092, filed on June 26, 1985, now U.S. Pat. No. 4,713,374, issuedDec. 15, 1987.

BACKGROUND AND FIELD OF THE INVENTION

The present invention relates to new functional ganglioside derivativesand more precisely to new esters and amides, their preparationprocedures, pharmaceutical preparations containing the gangliosideesters and amides and to the therapeutic use of the ganglioside estersand amides.

Gangliosides are natural products contained in various animal tissues ororgans, above all in the tissues of the central and peripheral nervoussystems, but also in the adrenal marrow, in the erythrocytes, in thespleen and elsewhere, from which they can be extracted in a purifiedform. It has been possible to establish the basic structure of mostgangliosides thus obtained. They are glycosphingolipids, that is,compounds resulting from the union of an oligosaccharide with asphingosine and a certain number of sialic acids bound together byglucosidic or ketosidic bonds. The gangliosides so far described in theliterature and obtained in purified form do not represent unitarychemical compounds, except possibly for their saccharide part(oligosaccharide), as the ceramide and sialic components are quitevariable, within certain limits. Thus, even when "pure" gangliosides arereferred to, this expression is to be broadly interpreted to mean aganglioside species in which at least a part, for instance thesaccharide part, is unitary and characteristic from a chemical point ofview. Given this, before describing in more detail the background of thepresent invention, it is useful to take note of the following generalformula which includes all the structures of the gangliosides so farobtained in purified form, and emphasizes the functions which arefunctionally modified according to the invention (formula I). ##STR1##In this formula, an oligosaccharide residue, formed by a maximum of 5monosaccharides, is connected by a glucoside bond to a ceramide residueand to one or more sialic acid residues, both by means of as many directglucoside bonds, and by means of one or more such bonds, as theremaining sialic acid residues are joined together by ketoside bonds.The formula shows the hydroxyl groups of the saccharide portion, of thesialic acids and of the ceramide, as well as the above mentionedglucoside bonds with sialic acids and ceramide and the carboxyl groupsof the sialic acids. The sialic acids which form part of thegangliosides of formula I have the general structure II ##STR2## inwhich one or more of the primary and secondary hydroxyl groups may alsobe acylated and in which the acyl groups derive from acetic or glycolicacid. The number of sialic acids present in gangliosides usually variesfrom 1 to 5.

The sialic residues are bound to the oligosaccharide by a ketosidic bondformed by the hydroxyl in position 2 with an oligosaccharide hydroxyl.When several sialic acids are bound together, their molecules are unitedby means of ketoside bonds formed among the hydroxyls of positions 2 and8 of two sialic acid molecules. The sialic acids of gangliosides,including purified gangliosides as described above, are mixtures ofvarious chemically unitary acids, such as N-acetylneuraminic andN-glycolylneuraminic acids, in which the first is predominant, andpossibly of one or more of their O-acylderivatives, such8-0-acylderivatives.

Gangliosides are to be found in nature as metallic salts, such as sodiumsalts, and it is therefore the carboxylic function/s of sialic acidswhich are salified. The free forms of gangliosides may be easilyobtained by treatment of the salts, such as sodium salts, which an acidtype ionic exchanger, using for example a resin such as Dowex AG 50×8,H⁺ form.

The ceramide residue in the gangliosides of formula I generallyrepresents several N-acyl sphingosine having one of the formulas##STR3## in which n=10-16 and the acyl derives from a saturated orunsaturated fatty acid having between 16 and 22 carbon atoms, or from acorresponding hydroxyacid.

The oligosaccharide is made up of a maximum of 5 monosaccharides ortheir derivatives with an acylaminic group, especially of hexoses andtheir derivatives of the above mentioned type. At least one glucose orgalactose molecule is, however, always present in the oligosaccharide.The most frequent residue present as an acylaminic derivative of theabove mentioned sugars is N-acetylgalactosamine or N-acetylglucosamine.

In order to better illustrate the structure of the gangliosides includedin formula I and in particular the character of the bonds between thesaccharide parts, the sialic acids and ceramide, reproduced below is theformula of a "pure" GM₁ ganglioside containing only one sialic acid(represented by N-acetylaminic or N-glycolylneuraminic acid). ##STR4##

It is well known that gangliosides are functionally important in thenervous system and it has recently been shown that gangliosides areuseful in the therapy of peripheral nervous system pathologies. Thetherapeutic action of gangliosides seems to consist above all instimulating sprouting phenomena of the nervous cell and in activatingthe membrane enzymes involved in the conduction of nervous stimuli, suchas the enzyme (Na⁺, K⁺) ATPase. Neuronal sprouting stimulated bygangliosides promotes functional recovery of the damaged nervous tissue.

Further studies have been carried out to find compounds which couldprove more efficient than gangliosides in the therapy of nervous systempathologies. These studies have led for example to the discovery thatinternal esters of gangliosides, in which one or more hydroxyls of thesaccharide part are esterified with one or more carboxylic groups of thesialic acids (intramolecular reaction) with the formation of as manylactonic rings, are more active than gangliosides themselves inpromoting neuronal sprouting and in activating membrane enzymes involvedin the conduction of nervous stimuli, such as the enyzme NA⁺, K⁺) ATPase(see U.S. Pat. No. 4,476,119).

According to the present invention, another group of gangliosidederivatives has now been discovered, which presents advantages overgangliosides themselves, inasmuch as they have a prolonged activity intime ("retard" effect). These compounds are derivatives in which thecarboxyl group of the sialic acids are functionally modified byesterification or by conversion into amides and derivatives of thoseesters or amides in which the hydroxyl groups of the saccharide part, ofthe sialic acids and of the ceramide are also esterified with organicacids, or rather acylate derivatives (which shall be simply called"acylates" hereinafter, and specifically "acetylates, propionylates,etc."). The derivatives of the invention also include gangliosidederivatives in which only the hydroxyl groups are esterified withorganic acids, that is, contain free carboxyl groups.

Two methyl esters of the carboxyl of a sialic acid of gangliosides weredescribed in the article "Notes on improved procedures for the chemicalmodification and degradation of glycosphyngolipids" in the Journal ofLipid Research 21, 642-645 (1980) by MacDonald et al. These compoundsare the methyl esters of the gangliosides G_(M1) and G_(M3) [(theabbreviations used herein to identify gangliosides are those proposed bySvennerholm in J. Jeurochem. 10, 613 (1963))]. However, MacDonald et al.do not report any biological activity for the compounds. The methylester of the ganglioside G_(M3) is also used in the preparation of oneof its peracylate derivatives for use in several degradation orcopulation reactions [Methods of Enzymology, 50, 137-140 (1978)]. Theabove mentioned article in Journal of Lipid Research by MacDonald et al.also describes an acetylation of the methyl esters of gangliosidesG_(M1) and G_(M3), but without isolating the acylated compounds.

Acetylation with acetic anhydride-pyridine of lipids extracted from thespleen, liver and kidneys of Buffalo rats, from Morris hepatomas andfrom fibroblast cells was described by Terunobo Saito and Sen-ItirohHakomori in the Journal of Lipid Research, 12, 257-259, (1971). Theauthors isolated by chromatography the acetylated mixture of thegangliosides contained in those lipids from the acylated product,together with other glycolipids without, however, identifying anyspecific ganglioside. Of the amides, the unsubstituted amide of theganglioside G_(M3) was described [Ad. Exp. Med. Biol. 19, 95 (1972)],but even in this case, no biological properties were mentioned.

OBJECTS AND SUMMARY OF THE INVENTION

One of the first objects of the present invention, therefore, resides inproviding the new functional ganglioside derivatives discussed above,that is, the esters and amides of the carboxyl groups of gangliosidesdefined in formula I, or of their mixtures, the peracylated derivativesof these esters and amides in the hydroxyl groups of theoligosaccharide, of sialic acids and ceramide of gangliosides of formulaI or of their mixtures with free carboxylic functions, and saltsthereof.

A second object of the invention resides in providing new pharmaceuticalpreparations containing both the new functional derivatives definedabove together with the well known ones described above.

A third object resides in the therapeutic use of all these gangliosidederivatives.

DETAILED DESCRIPTION OF THE INVENTION

A. The ganglioside starting compounds:

The present invention comprises new useful ganglioside derivatives.These new derivatives are particularly derived by functionally modifyingthe carboxyl and/or hydroxyl groups present in the basic gangliosidestructure. Gangliosides thus far obtained in purified form can berepresented by the following formula (I) which also emphasizes thefunctional groups modified according to the present invention: ##STR5##

These gangliosides which form the starting materials for the functionalderivatives of the invention are all those which may be extracted fromvarious animal organs and tissues, especially from the tissues of thecentral and peripheral nervous systems, for example from the brain,cerebrospinal fluid, muscles, plasma and blood serum, kidneys, adrenals,liver, spleen, intestine, and erythrocytes or leukocytes. The startinggangliosides may also be the purified ones described in literature, suchas those extracted from tissues and organs of vertebrates, especiallymammals such as man, cattle, calf, rat, mouse or from microorganisms.

According to the present invention, these ganglioside compounds aremodified by functionally modifying the hyroxyl and/or carboxyl groups inthe starting ganglioside molecule to produce new gangliosidederivatives. The new derivatives of the invention are particularlyobtained by (a) subjecting the carboxyl groups to esterification orconversion to an amide; and/or (b) acylating the hydroxyl groups presentin the ganglioside.

The esters or amides which are the new derivatives of the invention areparticularly monoesters and monoamides in the case ofmonosialogangliosides and polyesters and polyamides in the case ofpolysialogangliosides, with as many ester or amide groups as there arecarboxyl groups present in the molecule and, therefore, as many sialicacid groups as are present.

Ganglioside derivatives according to the invention can be prepared bymodifying "purified" individually characterized gangliosides or bymodifying a mixture of gangliosides, such as a mixture ofmonosialogangliosides and polysialogangliosides. In the mixtures whichare used for esterification or conversion into amides for thepreparation of active compounds according to the invention, such as forexample the mixture described in Example 3 hereinbelow, containing bothmonosialogangliosides and polysialogangliosides, all of the carboxylgroups are modified and derivatives are obtained which are totallyesterified or converted into amides. The description "esters or amides"utilized herein in this description should therefore be interpreted inthis sense to mean totally esterified or converted into amides. Thisalso applies especially for the derivatives of the illustrative Examplesgiven below which are simply referred to as "esters or amides". Thesestatements mean mixtures containing polysialogangliosides which aretotally esterified or converted in the amides in all of the carboxylgroups.

Thus, the present invention encompasses derivatives of gangliosideswhether derived from a single "purified" ganglioside or from a mixtureof gangliosides. The invention further encompasses derivatives obtainedfrom various ganglioside structures, particularly as the structure ofthe ganglioside may vary with respect to the number and kind of sialicacid residue, ceramide residue, or oligosaccharide residue.

Regarding their structure, the basic starting gangliosides may bemonosialo-, disialo-, trisialo-, tetrasialo-, or pentasialogangliosides,with the preferred sialic acids being N-acetylneuraminic andN-glycolylneuraminic acids. The sialic acids may also be acylated at oneof the hydroxyls of their lateral chain, such as the hydroxyl inposition 8, if this position is not already occupied by a ketosidic bondwhich binds it to another adjacent sialic residue.

The ceramide part may vary, in the manner discussed above, and also inthe length of the carbon atom chains of the sphingosines which comprisea part of the ceramide which may vary from 16 to 22 carbon atoms. Inaddition, the length of any acyl residue may also vary, particularlywithin the same limits of 16 to 22 carbon atoms. Apart from this, theceramide residue may vary in that the double sphingosine bond may beabsent or present and usually this residue is largely composed ofunsaturated N-acylated sphingosine and of a low percentage of thecorresponding saturated compound (which may however reach about 10%).The acyl group may also be derived from aliphatic hydroxyacids with theabove mentioned number of carbon atoms varying from 16 to 22. Oneparticularly important group of gangliosides contains, in the ceramideresidue, acylated sphingosines with 18 or 20 carbon atoms in theirchains and corresponding saturated compounds, while their saturated orunsaturated acyl group, unsubstituted by hydroxyls, has the same numberof 18 or 20 carbon atoms.

As discussed above, the present invention relates, on the one hand, tofunctional derivatives of "pure" gangliosides of formula I, that is witha unitary composition as described above, and, on the other hand, to thefunctional derivatives of ganglioside mixtures, for example, in theextract form as they are obtained from various animal tissues. In thefirst case the basic gangliosides are preferably those in which theoligosaccharide is formed by a maximum of 4 hexose residues orN-acetylhexosamine, since at least one hexose residue is present, and inwhich this saccharide part is chemically unitary. The hexoses arepreferably chosen from the group consisting of glucose and galactose andthe N-acetylhexosamines from the group comprising N-acetylglucosamineand N-acetylgalactosamine (ganglioside group A). The gangliosides inthis group are, for example, those extracted from the brains ofvertebrates, such as those described in the article "Gangliosides of theNervous System" in Glycolipid Methodology, Lloyd A. Witting Fd.,American Oil Chemists' Society, Champaign, Ill. 187-214 (1976) seeespecially plate 1), for example gangliosides G_(M4), G_(M3), G_(M2),G_(M1) -GlcNAc, G.sub. D2, G_(D1a) -GalNAc, G_(T1c), G_(Q), G_(T1) andespecially those in which the oligosaccharide contains at least oneglucose or galactose residue and one N-acetylglucosamine orN-acetylgalactosamine residue, especially the following (gangliosidegroup B) ##STR6## where Glc stands for glucose, GalNAC stands forN-acetylgalactosamine, Gal stands for galactose, and NANA stands forN-acetylneuraminic acid.

If ganglioside mixtures are used as the starting material for functionalconversions according to the present invention, the mixtures may consistof those directly obtained by extraction of gangliosides from variousanimal tissues as "total" ganglioside extracts or as various fractionsthereof. Such extracts are described in literature for example, in thearticles mentioned above or also in "Extraction and analysis ofmaterials containing lipidbound sialic acids" in Glycolipid Methodology,Lloyd A. Witting Fd., American Oil Chemists' Society, Champaign, Ill.159-186 (1976) and "Gangliosides of the Nervous System" from the samebook, pp. 187-214. Some of the most important mixtures to be usedaccording to the present invention are ganglioside extracts obtainedfrom tissues from the nervous system, in particular from the brain, andcontaining gangliosides G_(M1), G_(D1a), G_(D1b) and G_(T1b) alreadymentioned above. Mixtures of this type are for example those describedin Example 2.

B. Types of ganglioside derivatives of the invention:

Specified hereinafter are the specific alcohol, amide and acyl functionswhich are especially suitable for obtaining particularly interesting newcompounds according to the invention and these functional groups are tobe taken into consideration both for "pure" unitary gangliosides and formixtures, especially those listed here.

In each of the ganglioside groups mentioned above the carboxyl groups ofthe sialic residues are present according to one of the objects of thepresent invention in esterified form, or in the form of amides.

1. Esterification

The groups of esters in the new ganglioside derivatives derive inparticular from alcohols of the aliphatic series and especially fromthose with a maximum of 12 and especially 6 carbon atoms, or from thoseof the araliphatic series with preferably only one benzene ring,possibly substituted by 1-3 lower alkyl groups (C₁₋₄), for instancemethyl groups, and a maximum of 4 carbon atoms in the aliphatic chain,or alcohols of the alicyclic or aliphaticalicyclic series with only onecycloaliphatic ring and a maximum of 14 carbon atoms or of theheterocyclic series with a maximum of 12 and especially 6 carbon atomsand only one heterocyclic ring containing an atom are chosen from thegroup formed by N, O and S. The amide groups of the carboxylic functionsin the ganglioside derivatives of the present invention derive fromammonia or amines of any class having preferably a maximum of 12 carbonatoms.

The alcohols and amines mentioned above may be substituted orunsubstituted, especially by functions chosen from the group formed byhydroxyl, amine, alkoxyl groups with a maximum of 4 carbon atoms in thealkyl, carboxyl or carbylcoxy (such as carbonylmethoxy andcarbonylethoxy) groups with a maximum of 4 atoms in the alkyl,alkylamine or dialkylamine residue with a maximum of 4 carbon atoms inthe alkyls, and may be saturated or unsaturated, especially with onlyone double bond. The alcohols esterifying the carboxylic functions ofgangliosides according to the present invention may be monovalent orpolyvalent, especially bivalent. Of the alcohols of the aliphaticseries, those lower alcohols with a maximum of 6 carbon atoms arepreferred, such as methyl alcohol, ethyl alcohol, propyl and isopropylalcohol, normal butyl alcohol, isobutyl alcohol, tertiary butyl alcohol,and of the bivalent alcohols, ethyleneglycol and propyleneglycol. Of thealcohols in the araliphatic series, those with only one benzene residueare preferred, such as benzyl alcohol and phenethyl alcohol; of thealcohols of the alicyclic series, preferred are those with only onecycloaliphatic ring, such as cyclohexyl alcohol (cyclohexanol), orterpene alcohols, such as menthanol, carvomenthol, or one of theterpineols or terpinenols or piperitols. Of the alcohols in theheterocyclic series, tetrahydrofuranol or tetrahydropyranol arepreferred. For the esterification of the carboxylic ganglioside groups,substituted aliphatic alcohols may also be used, for example, with aminefunctions, like aminoalcohols, such as those with a maximum of 4 carbonatoms and especially aminoalcohols with dialkyl --(C₁₋₄) - amine groupssuch as diethylaminoethanol.

2. Preparation of amides:

The carboxylic functions converted to an amide group according to thepresent invention either derive from ammonia (and the amide is in thiscase the unsubstituted amide --CONH₂) or from primary or secondaryamines, especially from those containing a maximum of 12 carbon atoms.These amines may be of an aromatic, heterocyclic or alicyclic, nature,but are especially aliphatic. The major object of the present inventionis the carboxylic derivatives of aliphatic amines with a maximum of 12carbon atoms, and these amines may have open, straight or ramifiedchains or may be cyclic, such as the alkylamines derived from alkylswith between 1 and 6 carbon atoms, such as methylamine, ethylamine,ethylmethylamine, propylamine, butyl amine hexylamine, dimethylamine,diethylamine, diisopropylamine, dihexylamine, or benzylamine.

These amine groups may further be substituted by groups chosen from thegroup formed of aminic, alkylaminic or dialkylaminic groups with amaximum of 4 carbon atoms in the alkyl groups, or by hydroxyl or alkoxylgroups with a maximum of 4 carbon atoms in the alkyl groups, such asdimethylaminoethylamine, dimethylaminopropyl-1-amine and6-hydroxyhexyl-1-amine, or alkylenamines derived from alkylene groupswith straight chains with between 3 and 6 carbon atoms or correspondingchains substituted by 1 to 3 methyl groups, such as pyrrolidine,piperidine and azepine. The alkyl or alkylene groups of these aminesalso be interupted in the carbon atom chain or substituted by otherheteroatoms, in particular by nitrogen atoms, and the amides of theinvention are derived in this case from diamines, such asethylendiamine, trimethylendiamine, piperazine; or should the alkyl oralkylene groups be interrupted or substituted by oxygen or sulphur atomsthe amides represent aminoalcohol derivatives, such as aminoethanol oraminopropanol or are derivatives of morpholine or thiomorpholine. Thesegroups thus include e.g. alkylamines of the above said type, e.g.alkylamines and dialkylamines of the above type, e.g. of 1-6 carbonatoms, which are further substituted in the alkyl moieties by furtheraminic, alkylamino or dialkylamino groups with a maximum of 4 carbonatoms in the alkyl groups, or by hydroxyl or alkoxyl groups with amaximum of 4 carbon atoms in the alkyl groups, such asdimethylaminoethylamino, 3-dimethylamino-propyl-1-amino and6-hydroxy-hexyl-1 amino groups. The esters and amides specified above ofthe gangliosides of groups A and B previously mentioned, and of theirmixtures, are of special interest in the present invention.

3. Acylation

The invention also includes the peracylated derivatives in the hydroxylsof the saccharide part, of the sialic acids and ceramide of the estersand of the amides described here. In these derivatives the acyl groupsmay be derived from acids of the aliphatic, aromatic, araliphatic,alicyclic or heterocyclic series; preferably from acids of thealiphatic, aromatic, araliphatic, alicyclic or heterocyclic series witha maximum of 10 carbon atoms and especially 6 atoms, such as formic,acetic, propionic acid, the butyric and valerianic acids and caproic orcapric acid. The acyl groups may also be derived from acids for examplewith the same number of carbon atoms, but substituted. The acyl groupsmay particularly derive from hydroxyacids, such as lactic acid;aminoacids, such as glycine; or dibasic acids, such as succinic, malonicor maleic acids. Of the aromatic acids, those with only one benzene ringare preferred, particularly benzoic acid and its derivatives withmethyl, hydroxyl, amine or carboxyl groups, such as p-aminobenzoic acid,salicylic acid or phthalic acid. In the process of the invention, theseacids are reacted in the form of an anhydride thereof for reacting withthe hydroxyl group of an esterified or free carboxyl-group ganglioside.

The invention also includes peracylated derivatives of gangliosides andtheir mixtures described above, with, however, free carboxyl functions.For these derivatives too, those acylated derivatives deriving from theacids described above are particularly important. Regarding alsoperacylated derivatives with free or esterified carboxylic functions, orin the form of amides, the ganglioside derivatives or groups A and B areparticularly important, as are their mixtures, particularly those withacyl groups and those of the esters and amides previously mentioned.Therefore, one group of new ganglioside derivatives which isparticularly preferred is that comprising ganglioside esters and amidesand their peracylated derivatives in the hydroxyl groups as well as suchperacylated derivatives with carboxyl functions of the said gangliosidesin free form. In these derivatives the ester groups are derived fromalcohols formed by the group consisting of aliphatic alcohols with amaximum of 6 saturated carbon atoms, unsubstituted or substituted byhydroxyl, alkoxyl groups with a maximum of 4 carbon atoms, aminic,alkylaminic or dialkylaminic groups with a maximum of 4 carbon atoms inthe alkyl groups, carboxylic groups, carbalcoxylic groups with a maximumof 4 carbon atoms in the alkyl residue, and the corresponding alcoholswith a double bond at the most, and araliphatic alcohols with only onebenzene ring, unsubstituted or substituted by between 1 and 3 methylgroups, cycloaliphatic or aliphatic - cycloaliphatic alcohols with acyclohexane ring, unsubstituted or substituted by between 1 and 3 methylgroups and a maximum of 4 carbon atoms in the aliphatic part, andtetrahydrofuranol and tetrahydropyranol.

The amide groups in the most preferred derivatives are derived fromammonia or alkylamines, dialkylamine or alkyleneamines, with a maximumof 6 carbon atoms in the alkyl groups and between 4 and 8 carbon atomsin the alkylene groups and in which the alkyl or alkylene groups may beinterrupted in the carbon atom chain by heteroatoms chosen from thegroup formed by nitrogen, oxygen and sulphur, as the group may be iminic-NH in the case of the presence of a nitrogen atom substituted by analkyl with a maximum of 4 carbon atoms and/or may be substituted bygroups chosen from the group formed by aminic, alkylaminic ordialkylaminic groups with a maximum of 4 carbon atoms in the alkylgroups, or by hydroxyl or alkoxyl groups with a maximum of 4 carbonatoms in the alkyl groups, or by araliphatic amines with only onebenzene ring which may be substituted by a maximum of 3 methyl groupsand with a maximum of 4 carbon atoms in the aliphatic part.

The acyl groups esterifying the hydroxyls in these most preferredderivatives derive from aliphatic acids, saturated or unsaturated with amaximum of 6 carbon atoms, which may also be substituted by a functionchosen from the groups comprising hydroxyl, aminic and carboxyl groups,and their salts. This group of most preferred derivatives, andespecially that of the gangliosides of groups A and B mentioned aboveand also the derivatives of ganglioside mixture, of groups A and B forexample (with the functional groups specified here) are of particularinterest as ingredients for the pharmaceutical preparations according tothe present invention. Of the specific new compounds of the presentinvention, particularly important for pharmaceutical preparations, thefollowing derivatives are important:

the ethyl ester of ganglioside G_(M1)

the propyl ester of ganglioside G_(M1)

the isopropyl ester of ganglioside G_(M1)

the normal butyl ester of ganglioside G_(M1)

the isobutyl ester of ganglioside G_(M1)

the tertiary-butyl ester of ganglioside G_(M1)

the cyclohexyl ester of ganglioside G_(M1)

the esters corresponding to those listed here containing the gangliosideG_(D1b) in place of ganglioside G_(M1)

the esters listed above containing the ganglioside G_(D1a) in place ofganglioside G_(M1)

the esters listed here containing the ganglioside G_(T1b) in place ofganglioside G_(M1)

the peracetylates of the esters named above

the perpropionilates of the esters named above

the per-n-butyrrilates of the esters named above

the permaleinylates of the esters named above

the permalonylates of the esters named above

the persuccinylates of the esters named above

the peracetylates of gangliosides G_(M1), G_(D1b), G_(D1a), G_(T1b)

and the perpropionylates, the per-n-butyrrylates, the permalonylates,the persuccinylates and the permaleinylates of the same gangliosides

the amid of ganglioside G_(m1)

the amide of ganglioside G_(D1a)

the amide of ganglioside G_(D1b)

the amide of ganglioside G_(T1b)

the methylamide, the ethylamide, the propylamide of gangliosides G_(M1),G_(D1b), G_(D1a), G_(T1b) and also the amides of these gangliosidesderiving from dimethylamine, diethylamine, pyrrolidine, piperidine,piperazine, morpholine, thiomorpholine, the peracetylates, theperpropionylates, the per-n-butyrrilates, the permalonylates, thepermaleinylates and the persuccinylates of the amides mentioned justabove, the methyl, ethyl, propyl, isopropyl, tertiary butyl, benzyl,allyl, ethoxycarbonylmethyl esters of the ganglioside mixturescontaining G_(M1), G_(D1a), G_(D1b), G_(T1b), as principal gangliosidesand especially the mixture obtained according to the illustrated exampleNo. 2, unsubstituted amide, methylamide, ethylamide, benzylamide,isopropylamide, dimethylamide, diethylamide, dimethylaminopropylamide,dimethylaminoethylamide, ethanolamide of gangliosides containing G_(M1),G_(D1a), G_(D1b), G_(T1b), as principal gangliosides and especially themixture obtained according to illustrated example No. 2, theperaceylated, per-n-butyrrylated, perpropionylated, permaleinylated,permalonylated, persuccinylated derivatives of a ganglioside mixturecontaining G_(M1), G_(D1a), G_(D1b), G_(T1b) as principal gangliosidesand especially the mixture obtained according to illustrated Example No.2.

From the new compounds according to the present invention with freecarboxyl functions, such as peracylates of gangliosides, for instancethose of groups A and B, metallic salts may be prepared, which also formpart of the invention. Metallic salts may also be prepared from otherderivatives of the invention which possess a free acid function, such asperacylated esters or amides with bibasic acids. Furthermore, saltsobtained by acid addition of ganglioside derivatives, containing a freeamine function also form part of the invention, such as esters withaminoalcohols. Of the metallic salts, particularly preferred are thosewhich can be used in therapy, such as the salts of alkaline and alkalineearth metals, such as potassium, sodium, ammonium, calcium, magnesium,salts, or salts of earth metals such as aluminum, but also the saltswith organic bases, such as primary, secondary or tertiary aliphatic,aromatic or heterocyclic amines, such as methylamine, ethylamine,propylamine, piperidine, morpholine, ephedrine, furfurylamine, choline,ethylendiamine, aminoethanol.

Of the acids able to give salts with ganglioside derivatives by acidaddition according to the invention, particularly preferred are thehydracids, such as hydrochloric acid, bromhydric acid, the phosphoricacids, sulphuric acid, the lower aliphatic acids with a maximum of 7carbon atoms, such as formic, acetic or propionic, succinic or maleicacid. Unusable therapeutic acids or bases, such as picric acid, can beused for the purification of the new ganglioside derivatives and formpart of the invention. Due to the close connection between the newderivatives in free form and in the form of their salts, thisdescription of the invention is to be considered as encompassing bothforms, unless the contrary is expressly stated.

The new ganglioside esters and amides of this invention generallyrepresent amorphous, colourless, or greyish powders which can be quitesuccessfully dissolved in water and polar solvents, such as loweraliphatic alcohols, for instance, methyl, ethyl or propyl alcohol, oralso in ketones, such as acetone or in amides, such as dimethylformamideor in sulphoxides, such as dimethylsulphoxide, or ethers, such asdioxane or tetrahydrofurane. Solubility in water is, however,considerably reduced in the derivatives acylated at the hydroxyl groups,while it is increased in the organic solvents mentioned above. For themanufacture of the pharmaceutical preparations in the form of solutionsfor parenteral use, the most suitable solvents will be chosen each time,according to the more or less hydrophil or lipophil character of the newderivatives.

Methods of Preparation

The present invention also includes preparation methods for the newganglioside derivatives described above or for those already known.These methods are on the one hand the conventional, already knownmethods for the preparation of the new esters and amides of carboxylicacids and for the acylation of hydroxyl groups for the preparation ofthe new acyl derivatives, except for those methods which would have theeffect of altering the ganglioside base, such as those making use ofhighly acidic agents or those which in any case are carried out inalkaline or acid hydrolyzing conditions, or also those methods which maycause undesired alkylation of the hydroxyl groups of the saccharidepart. On the other hand the invention also includes a new preparationmethod for both new and known esters, starting with the internal estersof gangliosides as described hereafter.

As the invention includes the preparation of ganglioside derivativeswith esterified carboxyl functional groups or in the form of amides andalso the acylated derivatives in the hydroxyl groups of thesederivatives, on the one hand it is possible in this way to modify thecarboxyl functions both of free gangliosides, that is with free hydroxylfunctions, and of gangliosides with already acylated hydroxyl functions.On the other hand, it is possible to acylate the hydroxyl functions inalready esterified derivatives or in the amide derivatives. It is alsopossible, according to the invention, to acylate the hydroxyl functionsalone, leaving the carboxyl functions free. Therefore, according to thepresent invention, a ganglioside or one of its peracylated derivativesis esterified in the carboxyl groups, or these are converted to amides,or the hydroxyl groups of these ganglioside derivatives or thegangliosides with free carboxyl functions are acylated. Should it bedesired, salifiable compounds obtained are converted into their salts.

1. Esterification of carboxyl groups:

Of the methods known for the preparation of carboxylic esters,mentionable are those used for the preparation of the already knownmethyl esters of gangliosides G_(M1) and G_(M3) described in the abovementioned article in Journal of Lipid Research, 21, 642-645 (1980).According to this article, it is possible to obtain the esters of thecarboxyl groups of gangliosides by reacting the latter with an alcoholof which the ester is to be obtained in the presence of an ionicexchanger, for example, a resin such as Dowex 50. The yield is limiteddue to the simultaneous formation of internal esters and the longerreaction times (2-3 days).

The same method is used, for example, also in the above mentionedarticle in Methods of Enzymology, 50, 137-140 (1978) for the preparationof the methyl esters of G_(M3) ganglioside. This kind of partialesterification can clearly also be obtained albeit with an even loweryield, in the absence of resins. Apart from the Dowex 50 resin, otheracid ionic exchangers can also be used, having the function ofconverting gangliosides which, as already stated, are generally presentand especially in extracts in the form of salts, particularly sodiumsalts, in free gangliosides. This operation of conversion of gangliosidesalts into free gangliosides is suitable also for all the otherconventional methods described herein, for functionally modifying thecarboxylic function, such as those for the preparation of amides.However, the best method described in the above mentioned articleconsists of esterifying the carboxyl or carboxyls present ingangliosides by passing an alcoholic solution of the desired alcohol ona resin such as Dowex -50Wx8 (100-200 mesh H form) and treating theeluate dissolved in the same alcohol with the correspondingdiazomethane. In the specific case described, the esters of gangliosidesG_(M1) and G_(M3) were prepared by treatment in this way with methanoland diazomethane, giving a very good yield.

Another good preparation method of esters of ganglioside carboxylsconsists of treating a ganglioside metallic salt with an etherifyingagent. Salts of alkaline or alkaline earth metals are used, or also anyother metal salt. As an etherifying agent, those per se known inliterature may be used, especially the esters of various inorganicacids, or organic sulphonic acids, such as the hydracids, in other wordsthe hydrocarbon halogenides, such as methyl, ethyl iodide etc., or theneutral or acid sulphates of hydrocarbons, sulphites, carbonates,silicates, phosphites or hydrocarbon sulphonates, such as benzo orp-toluol-sulphonate of methyl or chlorosulphonate of methyl or ethyl.The reaction can be carried out in a suitable solvent, such as analcohol, preferably the one corresponding to the alkyl group which is tobe introduced into the carboxyl group, but nonpolar solvents may also beused, such as ketones, ethers, such as dioxane or dimethylsulphoxide.

A new method which is characteristic of the present invention for thepreparation of ganglioside esters, and which may be used both for thepreparation of the new gangliosides according to the invention, and alsofor the preparation of the already known esters, consists of treating aninternal ganglioside ester with a mixture of the desired alcohol withone of its corresponding alcoholates. The reaction can be carried out ata temperature corresponding to the boiling point of the alcohol. Lowertemperatures may also be used but in this case the reaction times arelonger. It is also possible, relinquishing however the very good yieldand short reaction times, to treat the internal ester with just therelevant alcohol, preferably at a temperature corresponding to boilingpoint of the same. The internal esters are described for example in theabove mentioned Belgian Pat. No. 894024 and U.S. Pat. No. 4,476,119.

As alcoholates it is preferable to use alkaline metal alcoholates,especially sodium alcoholate

2. Preparation of amides:

The new ganglioside amides according to the present invention can beprepared by per se known methods, especially by the following methods.

(a) reaction of the internal esters of gangliosides with ammonia or withamines.

(b) reaction of the carboxyl esters of gangliosides with ammonia or withamines.

(c) reaction of the ganglioside acids with the carboxyl groups activatedwith ammonia or amines.

Reaction (a), which has been described in the case of the preparation ofthe amide of ganglioside G_(M3) (see above) can be effected by directtreatment, with or without solvent, of the internal ganglioside esterwith ammonia or with the amine whose amide is to be prepared. Thereaction can be effected at quite low temperatures, such as from -5 to+10° C., but room temperature or higher is preferable, for instancebetween 30° and 120° C. As solvents, ketones, aromatic hydrocarbons,dimethylformamide, dimethylsulphoxide, dioxane, or tetrahydrofurane canbe used.

Reaction (b) is effected preferably in the conditions described for (a).Apart from the esters described for the present invention, other estersmay also be used, such as esters with phenols.

For activation of the carboxyl group in the reaction according to (c),methods per se known in peptide chemistry are used, avoiding thoseinvolving conditions which are too acid or basic which would causedisruption of the ganglioside molecule. If the starting gangliosides arein the form of sodium salts for example, it is advisable to first treatthe salt with an ion exchanging resin of the Dowex type, or another acidion exchanger. For example, it is possible to use the method ofcondensation in the presence of carbodiimides such asdicyclohexylcarbodiimide, benzylisopropylcarbodiimide orbenzylethylcarbodiimide, in the presence of 1-hydroxybenzotriazol orcondensation in the presence of N,N' -carbonyldiimidazol.

3. Acylation of the hydroxyl groups:

Acylation of the hydroxyl groups of the saccharide, sialic part and ofthe ceramide also occurs in a way which is per se already known, forexample by acylation with a halogenide or an anhydride of the acid beingused for acylation, preferably in the presence of a tertiary base, suchas pyridine or collidine. The reaction may take place at a lowtemperature, such as room temperature, leaving the acid derivative, suchas anhydride, to react for quite a long time, 12 to 24 hours forexample, or at a slightly higher temperature such as 50°-100° C., for afew hours.

The invention also includes modifications in the preparation proceduresof the new derivatives, in which a procedure is interrupted at any givenpoint or in which preparation is begun with an intermediate compound andthe remaining stages are carried out, or in which the starting productsare formed in situ.

EXAMPLE 1 METHYL ESTER OF THE GANGLIOSIDE G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aredissolved in 200 ml of an anhydrous mixture of methylene chloride andmethanol 4:1. 176 mg (3.27 mM) of sodium methylate dissolved in 50 ml ofanhydrous methanol are added and the mixture is refluxed for 2 hours. Atthe end of the reaction the mixture is neutralized with Dowex AG 50x8anhydrous resin (H⁺ form), the resin is separated by filtration andwashed with methanol and the solution is evaporated by drying. Theresidue is gathered in 50 ml of methylene chloride/methanol 1:1 and thereaction product is precipitated by pouring it into 250 ml of acetone.The raw product (4.9 g) is purified by preparative high pressurechromatography with 60 H Merck silica gel, using as solvent a mixture ofchloroform/methanol/isopropanol/ammonium carbonate at 2%1140:820:180:140. The pure fractions are gathered, evaporated by drying,redissolved in 15 ml of chloroform/methanol 1:1 and the product isprecipitated with 75 ml of acetone. This product represents the methylester of the ganglioside G_(M1). Yield 4.2 g.

IR spectroscopy carried out of KBr pellets shows the typical bond of theester at 1750 cm⁻¹. Chromotography on silica gel plates withchloroform/methanol/CaCl₂ at 0.3% 55:45:10 and determined with Ehrlichreagent (Rf 0.72) showed the product to be a unitary compound and freefrom the internal ester used as starting product (Rf 0.75) and from theganglioside G_(M1) (Rf 0.65). By treatment with an 0.1N solution of Na₂CO₃ at 60° for an hour, the ester bond is split, giving the primaryproduct, G_(M1).

EXAMPLE 2 PREPARATION OF A GANGLIOSIDE MIXTURE (GA MIXTURE) BYEXTRACTION FROM BOVINE BRAIN TISSUE AND OF THE CORRESPONDING MIXTURE OFINTERNAL ESTERS (TO BE USED IN THE VARIOUS SUBSEQUENT EXAMPLES

Bovine brain cortex, removed from the animal, is homogenized inphosphate buffer at pH 6.8; 6 volumes of tetradydrofuran are added andthe resulting mixture is centrifuged. The supernatant is re-extractedtwice with tetrahydrofuran. After contrifugation the non-polar materialsare removed by separation with ethyl ether and theaqueous-tetrahydrofuranic layer is introduced on an ionic exchangecolumn balanced with 50% ethanol. Barium hydroxide and four volumes ofice cold ethanol are added to the effluent from the column.

After 18 hours in cold conditions, the precipitate is gathered and thenslightly acidified with hydrochloric acid after solution in water. Thesolution thus obtained is dialyzed and freeze-dried. The yield at thispoint is of about 0.6 mg of raw ganglioside mixture per gram of nervoustissue used. The freeze-dried powder is dispered in 20 volumes ofchloroform-methanol 2:1, the solution obtained is filtered until it isperfectly clear, and then separated by adding 0.2 volumes of a solutionof potassium chloride in water at 0.88%.

The upper layer is separated, dialyzed and freeze-dried. The final yieldis of about 0.3 mg of purified mixture of ganglioside salts per gram ofbrain tissue.

5 grams of a mixture obtained according to the methods described aboveare dissolved in 50 ml of DMSO. 4 grams of styrene-type, anhydrous resin(sulphonic acid) (50-100 mesh, H+ form) are added to the mixture and theresulting system is agitated for 30 minutes at room temperature. Thistreatment with an ionic exchange resin transforms all salified carboxylgroups. Complete transformation is confirmed by a suitable method ofphysical analysis, such as atomic absorption. The resin is then filteredunder suction and the solution is treated with 1.5 g ofdicyclohexylcarbodiimide and left to rest for an hour. The precipitateddicyclohexylurea is removed by filtration and the resulting solution istreated with 100 ml of 0 causing precipitation of the internalganglioside esters produced.

The yield is of 4.6 g of mixture of internal esters (about 90-95% of thetheoretical value). The presence of internal ester derivatives isconfirmed by infrared spectroscopy and by then layer chromatography. IRSpectroscopy on KBr pellet: the esterifying lactonic bond produces aband at 1750 cm⁻¹. Thin layer chromatography: on silica gel plate,developiong solvent: CHCl₃ /MeOH/CaCl₂ O.3% (55:45:10, v/v/v), the Rf ofthe internal ester mixture is between 0.7 and 0.85. The Rf of the finalproducts is greater than the Rf of the mixture of starting substance;consequently chromatography shows the absence of starting material. Bytreatment with a solution of 0.1 N of Na₂ CO₃ at 60° for an hour theester bonds are split and it is possible to obtain the primary mixtureof the starting gangliosides.

The ganglioside mixture obtained can be fractioned in various portionssubstantially representing pure gangliosides (in the sense used in thegeneral description), using silicic acid columns and eluting with amixture of methanolchloroform. In this way an average composition isobtained of about 40% of the ganglioside G_(D1a), 21% of the gangliosideG_(M1), 19% of the ganglioside G_(T1b) and 16% of the gangliosideG_(D1b) .

EXAMPLE 3 MIXTURE OF METHYL ESTERS OF A GANGLIOSIDE MIXTURE

5 g of a mixture of internal esters of a mixture of gangliosides(obtained by extraction from bovine brain tissue as described in Example2) are dissolved in 200 ml of an anhydrous mixture of methylene chlorideand methanol 4:1. 318 mg (5.86 mM) of sodium methylate dissolved in 50ml of anhydrous methanol are added and the mixture is refluxed for 2hours. The raw product of the reaction is then isolated as described inExample 1 (4.9 g). The raw product is then purified by chromatography onSephadex-DEAE acetate form A-25, using as solvent a mixture ofchloroform/methanol/water 30:60:8. The mixed neutral fractions areevaporated, dialyzed in water, evaporated again by drying, the residuesare dissolved in 15 ml of chloroform/methanol 1:1 and the product isprecipitated with 75 ml of acetone. Yield: 4.3 g. IR spectroscopy,carried out on KBr pellets, showed the typical bond of the ester at 1750cm⁻¹. Chromatography carried out as described in Example 1 showed theproduct which represents the mixture of methyl esters of thegangliosides, to have an Rf of 0.72-0.85 (Rf of the ganglioside mixture,0.2-0.70).

Complete transesterification may be confirmed by determining themolecular proportion between the alkoxy and sialic groups which areobtained by quantitative "head space" gas-chromatography of the methylalcohol released after treatment with 0.1N solution of Na₂ CO₃ at 60°for one hour, causing the splitting of all the ester bonds, and withSvennerholm's method for determination of N-acetylneuraminic acid.

EXAMPLE 4 ETHYL ESTER OF THE GANGLIOSIDE G_(M1)

The ethyl ester of GM₁ is prepared and isolated in the same manner asfor the methyl ester in Example 1, using however ethyl alcohol andsodium ethylate in the place of methyl alcohol and sodium methylate, andusing the same molar quantities of internal ester and ethylate as inExample 1. Washing of the Dowex resin is effected with ethyl alcohol andthe residue obtained by evaporation of the filtered substance isdissolved in 50 ml of methylene/chloride ethanol 1:1. The yield in rawproduct is 4.9 g. Purification is also carried out as in Example 1.Yield of the ethyl ester of purified G_(M1) ganglioside: 4.3 g.Chromatographic analysis of the product carried out in the same way asin Example 1 shows the presence of a unitary compound with an Rf of0.80, and the absence of G_(M1) ganglioside and its internal ester (Rf0.65 and 0.75, respectively). Hydrolysis with NaCO₃ as described inExample 1 produces the ganglioside G_(M1). IR spectroscopic examinationon KBr pellets shows the typical band of the ester at 1750 cm⁻¹.

EXAMPLE 5 MIXTURE OF ETHYL ESTERS OF A MIXTURE OF GANGLIOSIDES

The mixture of ethyl esters is prepared and isolated in the same way asfor the mixture of methyl esters of Example 3, using however ethylalcohol and sodium ethylate instead of methyl alcohol and sodiummethylate, and using 5 g of internal ester mixture and 318 mg (5.86mM)of sodium ethylate.

Washing of the Dowex resin is carried out with ethyl alcohol and theresidue obtained by evaporation of the filtered substance is dissolvedin 50 ml of chloroform/ethanol 1:1. The yield in raw product is 4.9 g.Purification is also carrier out as in Example 3. The yield in purifiedethyl ester mixture: 4.5 g. IR spectroscopy, carried out on KBr pellets,shows the typical band of the ester at 1750 cm⁻¹. When it ischromatographed on silica gel plates with a freshly prepared solution ofchloroform/methanol/hydroxide of tetramethylammonium 1M 55:45:10 anddetermined with Ehrlich reagent, the product shows an Rf of 0.50-0.75(ganglioside mixture 0.20-0.60).

Complete transesterification is demonstrated in the same way asdescribed in Example 3.

EXAMPLE 6 ISOPROPYL ESTER OF THE GANGLIOSIDE G_(M1)

This derivative is prepared and isolated in the same way as for themethyl ester in Example 1, using however isopropyl alcohol and sodiumisopropylate instead of methyl alcohol and sodium methylate, and usingthe same molar quantities of internal ester and isopropylate as inExample 1. Washing of the Dowex resin is effected with isopropyl alcoholand the residue obtained by evaporation of the filtered substance isdissolved in 50 ml of methylene chloride/isopropanol 1:1. The yield inraw product is 4.9 g. Purification is also carried out as in Example 1.Yield in isopropyl ester of purified G_(M1) ganglioside: 4.2 g.

Chromotographic analysis of the product, carried out in the same way asin Example 1, shows the presence of a unitary compound with an Rf of0.85 and the absence of G_(M1) ganglioside or its internal ester.Hydrolysis with Na₂ CO₃ as described in Example 1 produces theganglioside G_(M1). IR spectroscopy on KBr pellets shows the typicalester band at 1750 cm-1.

EXAMPLE 7 MIXTURE OF ISOPROPYL ESTERS OF A GANGLIOSIDE MIXTURE

This derivative mixture is prepared and isolated in the same way as forthe mixture of methyl esters in Example 3, using however isopropylalcohol and sodium isopropylate instead of methyl alcohol and sodiummethylate, and using 5 g of internal ester mixture and 537 mg (6.52 mM)of sodium isopropylate. Washing of the Dowex resin is effected withisopropyl alcohol and the residue obtained by evaporation of thefiltered substance is dissolved in 50 ml of chloroform/isopropanol 1:L.The yield in raw product is 4.9 g. Purification is also carried out asin Example 3, using however as chromatographic eluent a mixture ofchloroform/isopropyl alcohol/water 20:60:8. Yield in mixture of purifiedisopropyl esters: 4.3 g.

Chromatographed by the method described in Example 5, the product showsan Rf of 0.40-0.78 (ganglioside mixture 0.20-0.60). IR spectroscopycarried out on KBr pellets, shows the typical ester band at 1750 cm⁻¹.Complete transesterification is demonstrated as described in Example 3.

EXAMPLE 8 TERTIARY BUTYL ESTER OF THE GANGLIOSIDE GM₁

This derivative is prepared and isolated in the same way as for themethyl ester in Example 1, using however tertiarybutyl alcohol andsodium tertiarybutylate instead of methyl alcohol and sodium methylate,and using the same molar quantities of internal esters andtertiarybutylate as in Example 1. Washing of the Dowex resin is effectedwith tertiarybutyl alcohol and the residue obtained by evaporation ofthe filtered substance is dissovled in 50 ml of methylenechloride/tertiarybutanol 1:1. The yield in raw product is 4.9 g. Yieldin tertiarybutyl ester of purified G_(M1) ganglioside: 4.1 g.

Chromatographic analysis of the product, carried out in the same way asin Example 1, shows the presence of a unitary compound with an Rf or0.71 and the absence of G_(M1) ganglioside and of its internal ester.Hydrolysis with Na₂ CO₃ as described in Example 1 produces theganglioside G_(M1). IR spectroscopy on KBr pellets shows the typicalester band at 1750 cm⁻¹.

EXAMPLE 9 MIXTURE OF TERTIARYBUTYL ESTERS OF A MIXTURE OF GANGLIOSIDES

This mixture is prepared and isolated in the same way as for the mixtureof methyl esters in Example 3, using however tertiarybutyl alcohol andsodium tertiarybutylate instead of methyl alcohol and sodium methylate,and using 5 g of internal ester mixture and 628.6 mg (6.52 mM) of sodiumtertiarybutylate. Washing of the Dowex resin is effected withtertiarybutyl alcohol and the residue obtained by evaporation of thefiltered substance is dissolved in 50 ml of chloroform/tertiarybutanol1:1. The yield in raw product is 4.9 g. Purification is also carried outas in Example 3, using however as chromatographic eluent a mixture ofchloroform/tertiarybutyl alcohol 1:1. Yield in purified tertiarybutylesters: 4.1 g. IR spectroscopy carried out on KBr pellets shows thetypical ester bond at 1750 cm⁻¹. Chromatographed by the method describedin Example 5, the product shows an Rf of 0.25-0.70. Complete reaction isdemonstrated as described in Example 3.

EXAMPLE 10 BENZYL ESTER OF THE GANGLIOSIDE G_(M1)

5 g of potassium salt of the ganglioside G_(M1) (3.14 mM) are dissovledin 50 ml of DMSO and 1.58 g (12.5 mM) of benzyl chloride and 2.08 g(12.5 mM) of KI are added to the solution. It is left to react innitrogen for 24 hours at 25° C. At the end of the reaction the solutionis partitioned with n-butanol/water 2:1 to eliminate the DMSO and thesalts. The butanol solution is evaporated by drying and the residue isgathered in 50 ml of chloroform/benzyl alcohol 1:1 and the reactionproduct is precipitated with 250 ml of acetone.

The raw product thus obtained (5.3 g) is then purified by preparativechromatography on silica gel plates using as solvent a mixture ofchloroform/methanol/water 65:32:7.

The pure fractions are mixed, evaporated, redissolved in 15 ml ofchloroform/isopropanol 1:1 and the product is precipitated with 75 ml ofacetone. Pure benzyl ester yield: 4.8 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterband at 1750 cm⁻¹ and UV spectroscopy examination, carried out inabsolute ethyl alcohol shows three maximums at 250, 255 e 261 nM.

Chromatographed on silica gel plates with chloroform/methanol/CaCl₂ at0.3% 60:35:8 and with chloroform/methanol/ammonia 2.5N 55:45:12 anddetermined with Ehrlich reagent, the product proves to be unitary withan Rf of 0.65 and 0.53 respectively and to be free from G_(M1) startingproduct (Rf 0.40 and 0.45 respectively).

Treatment with 0.1N solution of Na₂ CO₃ at 60° for an hour, causes thesplitting of the ester bond giving the starting products (G_(M1) andbenzyl alcohol).

EXAMPLE 11 MIXTURE OF BENZYL ESTERS OF A MIXTURE OF GANGLIOSIDES

5 g of a mixture of salified gangliosides (potassium salts) obtained byextraction from bovine brain tissue as described in Example 2 and bysubsequent substitution of sodium with potassium (ionic exchange) aredissolved in 100 ml of DMSO and 3.3 (26.0 mM) of benzyl chloride and 216g (13.0 mM) of KI are added to the solution. The mixture is left toreact in nitrogen for 48 hours at 25° C. At the end of the reaction thesolution is partitioned with n-butanol/H₂ O 2:1 to eliminate the DMSOand the salts. The butanol solution is evaporated by drying and theresidue is gathered in 50 ml of chloroform/benzyl alcohol 1:1 and theproduct is precipitated with 250 ml of acetone. The raw product thusobtained (5.6 g) is then purified by chromatography with Sephadex -DEAE,acetate form, using as solvent a mixture of chloroform/methanol/water30:60:8.

The eluted neutral fractions are mixed, freed of solvent by evaporationand the residue is gathered with 15 ml of chloroform/isopropanol 1:1 andthe mixture of purified benzyl esters is precipitated with 75 ml ofacetone. Yield 4.9 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterbond at 1750 cm⁻¹ and UV spectroscopy examination carried out inabsolute ethyl alcohol, shows three maximums at 250, 255 and 261 nM.

Chromatographed on silica gel plates with chloroform/methanol/CaCl₂ at0.3% 60:25:8 and with chloroform/methanol/ammonia 2.5N 55:45:10 anddetermined with Ehrlich reagent, the product proves to possess an Rfvarying between 0.40 and 0.70 and between 0.29 and 0.53 respectively(for the original ganglioside mixture 0.05-0.40 and 0.12-0.46respectively).

Complete reaction is demonstrated as described in Example 3.

EXAMPLE 12 ALLYL ESTER OF THE GANGLIOSIDE G_(M1)

5 g of potassium salt (3.14 mM) of the ganglioside G_(M1) are dissolvedin 50 ml of DMSO and 453.7 mg (3.75 mM) of allyl bromide and 625 mg(3.75 mM) of KI are added to the solution. It is left to react for 48hours at 25° C.

At the end of the reaction the solution is partitioned with n-butanol/H₂O 2:1 to eliminate the DMSO and the salts. The butanol solution isevaporated by drying and the residue is gathered in 50 ml ofchloroform/methanol 1:1 and the product is precipitated with 250 ml ofacetone.

The raw product thus obtained (5.1 g) is then purified by preparativecolumn chromatography on silica gel using as solvent a mixture ofchloroform/methanol/water 60:35:8.

The pure fractions are mixed, evaporated, redissolved in 15 ml ofchloroform/isopropanol 1:1 and the product is precipitated with 75 ml ofacetone. Pure allyl ester yield: 4.5 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterband at 1750 cm⁻¹.

Chromotographed on silica gel plates with chloroform/methanol/CaCl₂ at0.3% 60:40:9 with chloroform/methanol/ammonia 2.5N 55:45:10 anddetermined with Ehrlich reagent, the product proves to be a unitarycompound with Rf of 0.56 and 0.39 respectively and to be free fromstarting ganglioside G_(M1) (Rf 0.40 and 0.42 respectively).

Treatment with a solution of Na₂ CO₃ 0.1N at 60° for an hour causes thesplitting of the ester bond, giving starting ganglioside GM₁ and allylalcohol.

EXAMPLE 13 ETHOXYCARBONYLMETHYL ESTER OF THE GANGLIOSIDE GM₁

5 g of potassium salt (3.14 mM) of the ganglioside G_(M1) are dissolvedin 50 ml of DMSO and 2.12 g (12.5 mM) of ethylmonobromoacetate and 2.08g (12.5 mM) of KI are added to the solution. It is left to react innitrogen for 24 hours at 25° C. The solution is then partitioned withn-butanol/water 2:1 to eliminate the DMSO and the salts. The butanolsolution is evaporated by drying and the residue is gathered with 50 mlof chloroform/methanol 1:1 and the product is precipitated with 250 mlof acetone. Yield 4.8 g.

The raw product is purified by preparative column chromatography onsilica gel, using as solvent a mixture of chloroform/methanol/water60:32:7. The pure fractions are mixed, evaporated by drying, and theresidue dissolved in 15 ml of chloroform/methanol 1:1 and theethoxycarbonylmethyl ester is precipitated with 75 ml of acetone. Yield:2.4 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterband of 1750 cm⁻¹.

Chromatographed on silica gel plates with chloroform/methanol/CaCl₂ at0.3% 60:35:8 and determined with Ehrlich reagent, the product proves tobe a unitary compound with Rf of 0.64 and to be free from startingcompound G_(M1) (Rf 0.40).

Treatment with 0.1N solution of Na₂ CO₃ at 60° C. for an hour causessplitting of the ester bond, giving the original ganglioside G_(M1).

EXAMPLE 14 AMIDE OF THE GANGLIOSIDE G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aresuspended in 100 ml of anhydrous isopropyl alcohol. The suspension ismaintained in agitation at a low temperature (-5°) and dry ammonia isthen bubbled through it in anhydrous conditions for 3 hours.

At the end of the reaction, the solvent is eliminated by evaporation andthe residue is gathered in 50 ml of chloroform/methanol 1:1 and theproduct is precipitated with 250 ml of acetone.

The raw product (4.9 g) is treated with 100 ml of Na₂ CO₃ 1% for 30minutes at 25° C. to hydrolyze residue ester groups, dialyzed in water,evaporated by drying in vacuum and then purified by a preparative columnchromatography on silica gel, using as first solvent a mixture ofchloroform/methanol/H₂ O 60:40:9 and as second solvent a mixture ofchloroform/methanol/H₂ O 55:45:10. The pure, eluted mixed fractions areevaporated by drying, the residue is dissovled in 15 ml ofchloroform/methanol 1:1 and the amide is precipitated with 75 ml ofacetone. Yield: 4.8 g.

Chromatographed on silica gel plates with chloroform/methanol/ammonia 4N55:45:10 and chloroform/methanol/CaCl₂ at 0.3% 55:45:10 and determinedwith resorcinol reagent, the product proves to be a unitary compound (Rf0.10 and 0.32 respectively) and to be free from G_(M1) (Rf 0.35 and 0.65respectively).

EXAMPLE 15 MIXTURE OF THE AMIDES OF A GANGLIOSIDE MIXTURE

5 g of the mixture of internal esters of gangliosides described inExample 2 are reacted with ammonia as in Example 14 and precipitatedwith acetone also as described in Example 14. After a hydrolytictreatment with NaCO₃ as described in the previous Example, the rawproduct is purified as follows:

The product obtained by hydrolysis is dialyzed against water, thesolution is vacuum evaporated, the residue is gathered with 50 ml ofchloroform/methanol/H₂ O 30:60:8 and then purified by preparativechromatography with Sephadex DEAE A - 25, acetate form using as solventa mixture of chloroform/methanol/water 30:60:8.

The eluted neutral fractions are evaporated until dry, dialyzed,evaporated until dry once more, dissolved in 15 ml ofchloroform/methanol 1:1 and the mixture of amides is precipitated with75 ml of acetone. Yield: 4.8 g.

IR spectroscopy no longer shows the typical ester band at 1750 cm⁻¹.

Chromatographed on silica gel plates with chloroform/methanol/ammonia 4N55:45:10 and chloroform/methanol/CaCl₂ at 0.3% 55:45:10 and determinedwith resorcinol reagent, the product has an Rf varying between 0.01 and0.10 and between 0.55 and 0.45 respectively (mixture of originalganglioside, Rf 0.15-0.70 and 0.20-0.70 respectively).

EXAMPLE 16 METHYLAMIDE OF THE GANGLIOSIDE G_(M1)

5 g of internal ester of the ganglioside G_(M1) (3.27 mM) are suspendedin 25 ml of anhydrous methylamine in a recipient complete with refluxrefrigerator at -25° C. in anhydrous conditions. The suspension ismaintained in agitation at room temperature for 3 hours. At the end ofthe reacton the solvent is eliminated by evaporation and the residue isgathered with 50 ml of chloroform/methanol 1:1 and precipitated with 250ml of acetone.

The raw product thus obtained (4.9 g) is treated with 100 ml of Na₂ CO₃1% for 30 minutes at 25° C. to hydrolyze residue ester groups, and thendialyzed in water. The solution is evaporated until dry in vacuum andthe residue is purified by preparative chromatography with Sephadex DEAEA - 25, acetate form, using as solvent a mixture ofchloroform/methanol/water 30:60:8.

The mixed neutral fractions are evaporated until dry, dialyzed,evaporated again, dissolved in 15 ml chloroform/methanol 1:1 and themethylamide is precipitated with 75 ml of acetone. Yield: 4.8 g.

IR spectroscopy did not show the typical ester band at 1750 cm⁻¹.

Chromatographed on silica gel plates with chloroform/methanol/ammonia 4N55:45:10 and with chloroform/methanol/CaCl₂ at 0.3% 55:45:10 anddetermined with resorcinol reagent, the product proved to be a unitarycompound with Rf of 0.13 and 0.72 respectively and to be free fromG_(M1) (Rf 0.35 and 0.65 respectively).

EXAMPLE 17 MIXTURE OF METHYLAMIDES OF A GANGLIOSIDE MIXTURE

5 g of the internal ester mixture used in Example 15 is treated withmethylamine as in the previous Example and the reaction product istreated and isolated in the same way as in that Example. The yield ofpure product (mixture of methylamides of the ganglioside mixture used)is 4.8 g.

The Rf values determined as in the previous Example are 0.01-0.10 and0.20-0.45 respectively (mixture of original gangliosides: Rf 0.15-0.70and 0.20-0.70 respectively). The IR spectroscopic data are the same asthose in the previous Example.

EXAMPLE 18 ETHYLAMIDE OF THE GANGLIOSIDE G_(M1)

This derivative is prepared from 5 g of internal ester of theganglioside G_(M1) (3.27 mM) and from 25 ml of ethylamine in the sameway as in Example 16 and the same purification method is also followed.A yield of 4.8 g of pure ethylamide of the ganglioside G_(M1) isobtained.

The IR spectroscopic data are the same as for the methylamide in Example16 and chromatographic examination in the same conditions as in thatExample proved the product to be unitary and free from G_(M1) with Rf of0.19 and 0.75 respectively (Rf of G_(M1), 0.35 and 0.65 respectively).

EXAMPLE 19 MIXTURE OF ETHYLAMIDES OF A GANGLIOSIDE MIXTURE

This derivative mixture is prepared with 5 g of the mixture of internalesters of gangliosides used in Example 17 and 25 ml of ethylamine in thesame way as in Example 17, the same purification method is also used. Ayield of 4.8 g of the mixture of ethylamides of the ganglioside mixtureis obtained.

The Rf values determined by chromatography on silica gel plates withchloroform/methanol/ammonia 4N 55:45:10 and chloroform/methanol/CaCl₂ at0.3% 60:35:8 and determined with resorcinol reagent proved to be0.11-0.24 and 0.35-0.55 respectively (Rf of the original mixture ofgangliosides 0.15-0.70 and 0.05-0.40 respectively).

EXAMPLE 20 BUTYL-2-AMIDE OF THE GANGLIOSIDE G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aredissovled in 25 ml of anhydrous pyridine. 12.5 ml of 2-butylamine areadded to the solution and the mixture is kept in agitation in anhydrousconditions for 24 hours at 25° C.

At the end of the reaction the solvent is evaporated and the residuegathered with 50 ml of chloroform/methanol 1:1 and the reaction productis precipitated with 250 ml of acetone.

The raw product thus obtained (5.2 g) is then treated with 100 ml of Na₂CO₃ at 1% for 30 minutes at 25° C. in order to hydrolyze residue estergroups, dialyzed against water, the dialyzed solution is evaporated invacuum until dry and the residue is purified by preparativechromatography on silica gel, using as solvent chloroform/methanol/water110:40:6. The pure eluted fractions are mixed, the solution evaporateduntil dry, the residue dissolved in 15 ml of chloroform/isopropanol 1:1and the butylamide precipitated with 75 ml of acetone. The yield is 4.7g.

IR spectroscopy no longer shows the typical ester band at 1750 cm⁻¹.

Chromatography on silica gel plates with chloroform/methanol/ammonia 4N60:40:9 and chloroform/methanol/CaCl₂ at 0.3% 60:35:8 and determinedwith resorcinol reagent, showed the product to be unitary and free fromthe ganglioside G_(M1) with an Rf of 0.30 and 0.50 respectively (Rf ofG_(M1), 0.42 and 0.40 respectively).

EXAMPLE 21 BENZYLAMIDE OF THE GANGLIOSIDE G_(M1)

5 g of the internal ester (3.27 mM) of the ganglioside G_(M1) aredissolved in 20 ml of anhydrous pyridine and 396 mg (3.27 mM) ofbenzylamine are added to the solution which is then kept in agitation inanhydrous conditions for 24 hours at room temperature.

At the end of the reaction the solvent is eliminated by evaporation andthe residue is gathered with 50 ml of chloroform/methanol 1:1 and theproduct is precipitated with 250 ml of acetone.

The raw product thus obtained (5.1 g) is purified according to theprocedure described in Example 16, giving a yield of 4.6 g of the purebenzylamide ganglioside.

IR spectroscopy no longer shows the typical ester band at 1750 cm⁻¹.

Chromatography on silica gel plates with chloroform/methanol/ammonia 4N55:45:10 and chloroform/methanol/CaCl₂ at 0.3% 60:35:8 shows that theproduct is unitary and free from G_(M1) and presents Rf of 0.32 and 0.69(Rf of G_(M1) =0.35 and 0.40).

EXAMPLE 22 MIXTURE OF BENZYLAMIDES OF A GANGLIOSIDE MIXTURE

This mixture is prepared starting with 5 g of the ganglioside mixtureused in example 2 and with 792 mg (7.4 mM) of benzylamine according tothe procedure used in the previous Example for the preparation of thebenzylamide of the ganglioside G_(M1). Purification of the raw productobtained is also effected as in the previous Example. The yield is 4.8g.

IR spectroscopy and chromatography of silica gel plates are carried outas in the previous Example. The Rf values are 0.10-0.42 and 0.55-0.71respectively (primary ganglioside mixture, 0.01-0.15 and 0.05-0.40respectively).

IR spectroscopy no longer shows the typical ester band at 1750 cm⁻¹.

EXAMPLE 23 ISOPROPYLAMIDE OF THE GANGLIOSIDE G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aredissolved in 25 ml of anhydrous isopropylamine and the mixture is keptin agitation in anhydrous conditions for 24 hours.

At the end of the reaction the solvent is evaporated and the residue isgathered in 50 ml of chloroform/methanol 1:1 and the product isprecipitated with 250 ml of acetone.

The raw product (4.8 g) is treated with 100 ml of a solution of Na₂ CO₃at 1% for 30 minutes at 25° C. to hydrolyze residue ester bonds, andthen dialyzed in water. The dialyzed solution is evaporated until dryand purified by preparative chromatography on silica gel using assolvent a mixture of chloroform/methanol ammonia 2.5N 60:40:9. The pureeluted franctions are mixed, dissovled in 15 ml of chloroform/methanol1:1 and the product precipitated in 75 ml of acetone. Chromatography onsilica gel plates using as solvent chloroform/methanol/ammonia 2.5N60:40:9 and chloroform/methanol/CaCl₂ at 0.3% 60:35:8 and determinedwith resorcinol reagent, shows the isopropylamide (4.2 g) to be unitaryand free from G_(M1) and to have Rf of 0.25 and 0.66 respectively (Rf ofG_(M1), 0.42 and 0.40 respectively).

IR spectroscopy no longer shows the typical ester band at 1750 cm⁻¹.

EXAMPLE 24 DIMETHYLAMIDE OF THE GANGLIOSIDE G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aredissolved in 25 ml dimethylamine. The mixture is kept in agitation inanhydrous conditions at a low temperature (-5°) for 24 hours. At the endof the reaction the mixture is treated with Na₂ CO₃ and purified asdescribed in the previous Example, using however as first solvent forthe chromatography chloroform/methanol/ammonia 2.5N 60:40:9 and assecond solvent chlorofrom/methanol/water 60:40:9. The dimethylamideweights 4.6 g.

Spectroscopic and chromatographic examinations are carried out asdescribed in the previous Example. The product proves to be unitary withRf of 0.20 and 0.46, respectively, and free from G_(M1) (Rf 0.42 and0.40 respectively). IR spectroscopy shows no ester band at 1750 cm⁻¹.

EXAMPLE 25 MIXTURE OF DIMETHYLAMIDES OF A GANGLIOSIDE MIXTURE

This mixture is prepared by starting with 5 g of the mixture of internalesters of the gangliosides described in Example 2 and with 20 ml ofanhydrous dimethylamine according to the method in the previous Example.The subsequent treatment is also carried out as in the previous Example,except for the preparative chromatography, which is carried out onSephadex A-25, acetate form, using as solvent a mixture ofchloroform/methanol/water 30:60:8. The pure product weights 4.9 g.

IR spectroscopy and chromatography are effected as in the previousExample.

Rf values are 0.15-0.50 and 0.40-0.56 respectively (original gangliosidemixture, 0.15-0.60 and 0.05-0.40 respectively).

EXAMPLE 26 DIETHYLAMIDE OF THE GANGLIOSIDE G_(M1)

This compound is prepared in the same manner as the dimethylamide inExample 24, starting with 5 g of internal ester of the ganglioside GM₁and with 25 ml of anhydrous diethylamine. Purification is carried out inthe same manner as in Example 23. Yield: 4.7 g. Chromatographicexamination carried out as in Example 23 shows the product to be unitarywith Rf of 0.29 and 0.50 respectively and to be free from G_(M1). IRspectroscopy shows no ester band at 1750 cm⁻¹.

EXAMPLE 27 MIXTURE OF DIETHYLAMIDES OF A GANGLIOSIDE MIXTURE

This mixture is prepared by starting with 5 g of a mixture ofgangliosides of the internal esters used in Example 2 and 20 ml ofanhydrous diethylamine according to the method in the previous Example.Purification is effected as for the dimethylamide of the mixture inExample 25. Yield: 5.0 g.

Rf values (determined as in the previous Example) are 0.18-0.55 and0.43-0.60 respectively). IR spectroscopy shows no ester band at 1750cm⁻¹.

EXAMPLE 28 ETHYLMETHYLAMIDE OF THE GANGLIOSIDE G_(M1)

This compound is prepared in the same manner as in Example 24, startingwith 5 g of G_(M1) ganglioside and 25 ml of ethylmethylamine.Purification is also carried out as in the aforesaid Example. Yield: 4.7g.

Chromatography on silica gel plates is effected as in Example 24 and theproduct proves to be unitary and free from G_(M1). Rf=0.25 and 0.48respectively. IR spectroscopy shows no typical ester band at 1750 cm⁻¹.

EXAMPLE 29 3-DIMETHYLAMINOPROPYL-1-AMIDE OF THE GANGLIOSIDE G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aredissolved in 20 ml of anhydrous pyridine and 675 mg (6.6 mM) of3-dimethylaminopropyl-1-amine are added to the solution. The mixture isagitated at room temperature for 24 hours. The product is isolated as inthe previous Example and purified as in Example 28. Yield of3-diamethylaminopropyl-1-amide: 4.9 g.

From chromatography on silica gel plates, using as solventchloroform/methanol/ammonia 2.5N 55:45:10 and chloroform/methanol/CaCl₂at 0.3% 55:45:10 and determined with resorcinol reagent, the productproves to be unitary and free from G_(M1) and to have Rf of 0.02 and0.06 respectively (Rf of G_(M1), 0.45 and 0.65 respectively).

EXAMPLE 30 Maleate of 3-dimethylaminopropyl-1-amide of the gangliosideG_(M1)

5 g of 3-dimethylaminopropyl-1-amide of the ganglioside G_(M1) obtained,for example, as in the previous Example are dissolved in 100 ml ofchloroform/methanol 1:1 and 400 mg (3.44 mM) of maleic acid are added tothe solution.

After an hour of agitation at room temperature, the solution isevaporated in vacuum and the reside is gathered in 25 ml ofchloroform/methanol 1:1 and the product precipitated in 200 ml ofacetone. Yield in maleate of 3-dimethylaminopropyl-1-amide, 5.2 g.

EXAMPLE 31 Mixture of 3-dimethylaminopropyl-1-amides of of a gangliosidemixture

5 g of a mixture of the internal esters of gangliosides as described inExample 2 are dissolved in 20 ml of anhydrous pyridine and 1.51 g (14.8mM) of 3-dimethylaminopropyl-1-amine are added to the solution and themixture is agitated in anhydrous conditions for 24 hours at roomtemperature. Subsequent treatment is carried out as in Example 29. Yieldof 3-dimethylaminopropyl-1-amide of the purified ganglioside mixture is5.1 g.

Chromatography on silica gel plates, carried out as in Example 29 givesthe following Rf values: 0.01-0.05 and 0.01-0.10 respectively (primaryganglioside mixture, Rf 0.15-0.70 and 0.20-0.70 respectively).

EXAMPLE 32 Maleate of the mixture of 3-dimethylaminopropyl-1-amides ofganglioside mixture

5 of the mixture of 3-dimethylaminopropyl-1-amides of the gangliosidemixture, prepared as in the previous Example, are dissolved in 100 ml ofchloroform/methanol 1:1 and 697 mg (6 mM) of maleic acid are added tothe solution. After an hour of reaction, under agitation and at roomtemperature, the solution is evaporated in vacuum until dry and theresidues is gathered with 25 ml of chloroform/methanol 1:1 and theproduct precipitated in 200 ml of acetone. Yield in maleic salt, 5.3 g.

EXAMPLE 33 Dimethylaminoethylamide of the ganglioside G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aredissolved in 50 ml of an anhydrous solution of chloroform/isopropanol1:1 and then 537 mg (6.5 mM) of dimethylaminoethylamine are added.

The solution is agitated in anhydrous conditions for 24 hours at roomtemperature. Isolation and purification of the raw product thus obtainedare effected as in Example 31, giving a yield of 4.9 g.

The product, chromatographed in the conditions described in the previousExample proves to be unitary and to have Rf of 0.11 and 0.14respectively. IR spectroscopy shows no band at 1750 cm⁻¹.

EXAMPLE 34 Maleate of dimethylaminoethylamide of the ganglioside G_(M1)

5 g of dimethylaminoethylamide of the ganglioside G_(M1), obtained asdescribed in the previous Example, are dissolved in 100 ml ofchloroform/methanol 1:1 and 400 mg (3.44 mM) of maleic acid are added tothe solution. After an hour at room temperature under agitation, thesolution is evaporated in vacuum and the residue is gathered with 25 mlof chloroform/methanol 1:1 and the product precipitated in 200 ml ofacetone. Yield: 5.2 g.

EXAMPLE 35 Mixture of dimethylaminoethylamides of a ganglioside mixture

5 g of a mixture of internal esters of a ganglioside mixture asdescribed in Example 2, are dissolved in 50 ml of an anhydrous solutionof chloroform/isopropanol 1:1 and 955 mg (0.8 mM) ofdimethylaminoethylamine are added to the solution. The solution is keptat room temperature under agitation in anhydrous conditions for 24hours.

Subsequent isolation and purification of the raw product are effected asdescribed in Example 33. Chromatography on silica gel plates, carriedout as in Example 33, gives Rf values of 0.01-0.14 and 0.01-0.16respectively (original ganglioside mixture, 0.15-0.70 and 0.20-0.70respectively). IR spectroscopy shows no ester band at 1750 cm⁻¹.

EXAMPLE 36 Maleate of the mixture of dimethylaminoethylamides of theganglioside mixture

5 g of the mixture of dimethylaminoethylamides of the gangliosidesdescribed in the previous Example are dissolved in 100 ml ofchloroform/methanol 1:1 and 697 mg (6 mM) of maleic acid are added tothe solution.

After one hour, during which the solution is agitated at roomtemperature, the mixture is evaporated in vacuum until dry and theresidue is gathered in 25 ml of chloroform/methanol 1:1 and the productprecipitated with 200 ml of acetone. Yield: 5.3 g of the maleate.

EXAMPLE 37 Ethanolamide of the ganglioside G_(M1)

5 g of the internal ester of the ganglioside G_(M1) (3.27 mM) aredissolved in 50 ml of an anhydrous solution of chloroform/isopropanol1:1 and 397.2 mg (6.5 mM) of ethanolamine are added to the solution. Themixture is kept in anhydrous conditions under agitation at roomtemperature.

Isolation and purification of the reaction product are carried out as inExample 33, giving 4.9 g of pure product.

Chromatography on silica gel plates with chloroform/methanol/ammonia 4N55:45:10 and chloroform/methanol/CaCl₂ 0.3% 60:35:8 and determined withresorcinol reagent shows the product to be unitary and free G_(M1), withRf of 0.12 and 0.49 respectively.

EXAMPLE 38 Mixture of ethanolamides of a ganglioside mixture

This mixture is prepared with 5 g of the mixture of internal esters ofthe ganglioside mixture described in Example 2, dissolved in 20 ml ofanhydrous pyridine, and with 671 mg (10.8 mM) of ethanolamine. Themixture is agitated for 24 hours at room temperature.

Isolation of the raw product of the reaction, and its purification arecarried out as in the previous Example, giving a yield of 5.2 g.

Chromatography on silica gel plates, carried out as in Example 33, showsRf values of 0.09-0.56 and 0.25-0.55 respectively. IR spectroscopy didnot show any typical ester band.

EXAMPLE 39 6-hydroxyhexyl-1-amide of the ganglioside G_(M1)

5 g of internal ester of the ganglioside G_(M1) (3.27 mM) are dissolvedin 20 ml of anhydrous pyridine, and 762 mg (6.5 mM) of6-hydroxyhexyl-1-amine. It is left to react under agitation for 24 hoursat room temperature in anhydrous conditions. Isolation of the rawproduct of the reaction is carried out as in Example 28. Hydrolysis ofthe residue ester groups and dialysis are also carried out as in Example12 and the purified product is precipitated with acetone as in theprevious Examples. Yield: 4.3 g.

Chromatography on silica gel plates, using as solventschloroform/methanol/ammonia 2.5N 60:40:9 and chloroform/methanol/CaCl₂at 0.3% 60:35:8 and determined with resorcinol reagent, showed theproduct to be unitary and free from G_(M1), with Rf of 0.40 and 0.80respectively (Rf of G_(M1), 0.42 and 0.40 respectively).

EXAMPLE 40 Peracetylated derivative of the ganglioside G_(M1)

5 g of the sodium salt (3.19 mM) of the ganglioside G_(M1) are dissolvedin 50 ml of anhydrous pyridine and 25 ml of freshly distilled aceticanhydride are added to the solution at 25° C.

The solution is then kep in agitation for 72 hours at room temperature.At the end of the reaction, the solution is evaporated in vacuum untildry and the residue is partitioned with 100 ml of ice cold water and 200ml of ethyl acetate.

The ethyl acetate is then washed with cold HCl 1.0 M, with water andsolutions of NaHCO₃ 1.0 M. The organic layers are then anhydrified withsodium sulphate, evaporated in vacuum and the residue is purified bypreparative column chromatography on silica gel using a mixture ofdichloromethane/ethyl acetate/isopropanol 70:30:7 as eluent solvent. Thepure fractions are mixed. Evaporated until dry, redissolved in 20 ml ofethyl acetate and the product precipitated in 100 ml of normal hexane.

Chromatography on silica gel plates, using dichloromethane/ethylacetate/methanol 70:30:10 and ethyl acetate/isopropanol 95:5 anddetermined with Ehrlich reagent, shows the product to be unitary with Rfof 0.47 and 0.28 respectively.

EXAMPLE 41

Peracetylate derivative of a ganglioside mixture

5 g of the ganglioside mixture described in Example 2 in the form oftheir sodium salts, are dissolved in 25 ml of anhydrous pyridine and 25ml of acetic anhydride are added to the solution. The mixture is keptunder agitation for 72 hours at room temperature. At the end of thereaction the solution is evaporated in vacuum until dry and the residueis divided between 100 ml of ice cold water and 200 ml of ethyl acetate,the ethyl acetate is washed with cold HCl 1.0N, with water and with asolution of NaHCO₃ 1.0 M.

The organic layers are then anhydrified with sodium sulphate, evaporatedin vacuum and the residue gathered in 20 ml of ethyl acetate and theproduct precipitated in 100 ml of normal hexane. Yield: 4.4 g.

Chromatography carried out as in the previous Example gives the mixturean Rf of 0.01-0.46 and 0.01-0.36 respectively.

EXAMPLE 42 Peracetylated derivative of the methyl ester of theganglioside G_(M1)

5 g (3.17 mM) of the methyl ester of the ganglioside G_(M1) aredissolved in 50 ml of anhydrous pyridine and at 25° C., 25 ml of freshlydistilled acetic anhydride are added to the solution and the mixture iskept in agitation for 72 hours at room temperature. At the end of thereaction the solution is evaporated in vacuum and the residue dividedbetween 100 ml of ice cold water and 200 ml of ethyl acetate. The ethylacetate is washed with cold HCl 1.0 M, with water and with a solution ofNaHCO₃ 1 M. The organic layers are anhydrified with sodium sulphate,evaporated in vacuum and the residue purified by preparativechromatography on silica gel column, using as solvent a mixture ofdichloromethane/ethyl acetate/isopropanol 70:30:45.

The pure fractions are mixed, evaporated, redissolved in 20 ml of ethylether and precipitated in 100 ml of normal hexane. Yield 4.5 g ofperacetylated derivative of the methyl ester of G_(M1) ganglioside.Chromatography on silica gel plates with dichloromethane/ethylacetate/methanol 70:30:10 and ethyl acetate/isopropanol 95:5 anddetermined with Ehrlich reagent shows the product to be unitary with Rfof 0.47 and 0.28 respectively.

EXAMPLE 43 Peracetylated derivative of the mixture of methyl esters of aganglioside mixture

5 g of the mixture of methyl esters of the ganglioside mixture describedin Example 2 (see also Example 3) are acetylated as described in Example40. Purification of the product of the acetylation is also carried outas in Example 40, giving a yield of 5.4 of the peracetylated derivativeof the methyl ester mixture of Example 3.

Chromatography carried out as in the previous Example shows Rf in therange of 0.23-0.54 and 0.01-0.52.

EXAMPLE 44 Peracetylated derivative of the amide of the gangliosideG_(M1)

Starting with 5 g (3.20 mM) of the amide of G_(M1) ganglioside in 50 mlof anhydrous pyridine the acetylated derivative is prepared as inExample 42. Purification is carried out as in Example 41, using however,for the chromatography, dichloromethane/isopropanol 95:5 as solvent.Yield: 4.4 g of pure peracetylated derivative of the amide of G_(M1)ganglioside.

The product proves to be unitary when chromatographed as in the previousExample with Rf of 0.43 and 0.48 respectively.

EXAMPLE 45 Peracetylated derivative of the amide mixture of aganglioside mixture

5 g of the mixture of ganglioside amides described in Example 15dissolved in 50 ml of anhydrous pyridine are acetylated with 25 ml ofacetic anhydride as in the previous Example, Purification is carried outas in Example 42, giving 4.9 g of peracetylated derivative of theganglioside amides of Example 15.

Chromatographed in the same conditions as in the previous Example, thecompound has Rf of 0.11-0.45 and 0.01-0.50 respectively.

EXAMPLE 46 Phenylethyl ester of the ganglioside G_(M1)

The phenylethyl ester of GM₁ is prepared and isolated in the same manneras for the methyl ester in Example 1, using however phenethyl alcoholand sodium phenylethylate in the place of methyl alcohol and sodiummethylate and heating on a water bath, and using the same molarquantities of internal ester and sodium phenylethylate as in Example 1.Washing of the Dowex resin is effected with phenylethyl alcohol and theresidue obtained by evaporation of the filtered substance is dissolvedin 50 ml of methylenechloride/phenethyl alcohol 1:1. The yield in rawproduct is 5.1 g. Purification is also carried out as in Example 1.Yield of the purified phenylethyl ester of GM₁ ganglioside: 4.3 g.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.90 and the absence of GM₁ ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na2CO3as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 47 Mixture of phenylethyl esters of a mixture of ganglioside

The mixture of phenylethyl esters is prepared and isolated in the sameway as for the mixture of methyl esters of Example 2, using howeverphenethyl alcohol and sodium phenylethylate instead of methyl alcoholand sodium methylate and heating on a water bath, and using 5 g ofinternal ester mixture and 844.8 mg (5.86 mM) of sodium phenylethylate.Washing of the Dowex resin is carried out with phenylethyl alcohol andthe residue obtained by evaporation of the filtered substance isdissolved in 50 ml of chloroform/phenylethyl alcohol 1:1. The yield inraw product is 5.3 g. Purification is also carried out as in Example 3.The yield in purified ester mixture: 4.4 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 19M 55:45:10 and determined with Ehrlich reagent,the product shows an Rf of 0.65-0.887 (ganglioside mixture 0.2-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 48 2-Cyclohexylethyl ester of the ganglioside GM₁

The 2-cyclohexylethyl ester of GM₁ is prepared and isolated in the samemanner as for the methyl ester in Example 1, using however2-cyclohexylethanol and sodium 2-cyclohexyethylate in the place ofmethyl alcohol and sodium methylate and heating on a water bath, andusing the same molar quantities of internal ester and sodium2-cyclohexylethylate as in Example 1. Washing of the Dowex resin iseffected with 2-cyclohexylethyl alcohol and the residue obtained byevaporation of the filtered substance is dissolved in 50 ml ofmethylenechloride/2-cyclohexylethanol 1:1. The yield in raw product is5.1 g. Purification is also carried out as in Example 1. Yield of thepurified 2-cyclohexylethyl ester of GM₁ ganglioside: 4.3 g

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.92 and the absence of GM₁ ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na₂ CO₃as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 49 Mixture of 2-cyclohexylethyl esters of a mixture ofgangliosides

The mixture of 2-cyclohexylethyl esters is prepared and isolated in thesame way as for the mixture of methyl esters of Example 2, using however2-cyclohexylethanol and sodium 2-cyclohexylethylate instead of methylalcohol and sodium methylate and heating on a water bath, and using 5 gof internal ester mixture and 880.3 mg (5.86 mM) of sodium2-cyclohexylethylate. Washing of the Dowex resin is carried out with2-cyclohexylethyl alcohol and the residue obtained by evaporation of thefiltered substance is dissolved in 50 ml of chloroform/ethanol 1:1. Theyield in raw product is 5.3 g. Purification is also carried out as inExample 3. The yield in purified ester mixture: 4.3 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.67-0.90 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 50 Menthyl ester of the ganglioside GM₁

The menthyl ester of GM₁ is prepared and isolated in the same manner asfor the methyl ester in Example 1, using however menthol and sodiummenthylate in the place of methyl alcohol and sodium methylate andheating on a water bath, and using the same molar quantities of internalester and sodium methylate as in Example 1. Washing of the Dowex resinis effected with methyl alcohol/methylene chloride 1:1 and the residueobtained by evaporation of the filtered substance is dissolved in 50 mlof methylenechloride/menthol 1:1. The yield in raw product is 3.9 g.Purification is also carried out as in Example 1. Yield of the purifiedmenthyl ester of GM₁ ganglioside: 4.2 g.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.93 and the absence of GM₁ ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively. Hydrolysis with Na₂ CO₃as described in Example 1 produces the ganglioside GM₁.

Example 51 Mixture of menthyl esters of a mixture of gangliosides

The mixture of menthyl esters is prepared and isolated in the same wayas for the mixture of methyl esters of Example 2, using however mentholand sodium menthylate instead of methyl alcohol and sodium methylate andheating on a water bath, and using 5 g of internal ester mixture and1038.8 mg (5.86 mM) of sodium menthylate. Washing of the Dowex resin iscarried out with menthyl alcohol/methylene chloride 1:1 and the residueobtained by evaporation of the filtered substance is dissolved in 50 mlof chloroform/ethanol 1:1. The yield in raw product is 5.2 g.Purification is also carried out as in Example 3. The yield in purifiedester mixture: 4.3 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.70-0.93 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 52 Tetrahydrofurfuryl ester of the ganglioside GM₁

The tetrahydrofurfuryl ester of GM₁ is prepared and isolated in the samemanner as for the methyl ester in Example 1, using however,tetrahydrofurfuryl and sodium tetrahydrofurylate in the place of methylalcohol and sodium methylate and heating on a water bath, and using thesame molar quantities of internal ester and sodium tetrahydrofurfurylateas in Example 1. Washing of the Dowex resin is effected withtetrahydrofurfuryl alcohol and the residue obtained by evaporation ofthe filtered substance is dissolved in 50 ml of methylenechloride/tetrahydrofurfuryl alcohol 1:1. The yield in raw product is 5.0g. Purification is also carried out as in Example 1. Yield of thepurified tetrahydrofurfuryl ester of GM₁ ganglioside: 4.2 g.

IR spectroscopic examination of KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.72 and the absence of GM₁ ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na₂ CO₂as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 53 Mixture of tetrahydrofurfuryl esters of a mixture ofgangliosides

The mixture of tetrahydrofurfuryl esters is prepared and isolated in thesame way as for the mixture of methyl esters of Example 2, using howevertetrahydrofurfuryl alcohol and sodium tetrahydrofurfurylate instead ofmethyl alcohol and sodium methylate and heating on a water bath, andusing 5 g of internal ester mixture and 727.5 mg (5.86 mM) of sodiumtetrahydrofurfurylate. Washing of the Dowex resin is carried out withtetrahydrofurfuryl alcohol and the residue obtained by evaporation ofthe filtered substance is dissolved in 50 ml of chloroform/ethanol 1:1.The yield in raw product is 5.2 g. Purification is also carried out asin Example 2. The yield in purified ester mixture: 4.2 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.45-0.68 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 54 Tetrahydro-2H-pyran-4-yl ester of the ganglioside GM₁

The tetrahydro-2H-pyran-4-yl ester of GM₁ is prepared and isolated inthe same manner as for the methyl ester in Example 1, using howevertetrahydro-2H-pyran-4-ol and sodium tetrahydro-2H-pyran-4-ylate in theplace of methyl alcohol and sodium methylate and heating on a waterbath, and using the same molar quantities of internal ester and sodiumtetrahydro-2H-pyran-4-ylate as in Example 1. Washing of the Dowex resinis effected with tetrahydro-2H-pyran-4-ol and the residue obtained byevaporation of the filtered substance is dissolved in 50 ml of methylenechloride/tetrahydro-2H-pyran-4-ol 1:1. The yield in raw product is 5.1g. Purification is also carried out as in Example 1. Yield of thepurified tetrahydro-2H-pyran-4-yl ester of GM₁ ganglioside: 4.3 g.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.73 and the absence of GM₁ ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na₂ CO₃as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 55 Mixture of tetrahydro-2H-pyran-4-yl esters of a mixture ofgangliosides

The mixture of tetrahydro-2H-pyran-4-yl esters is prepared and isolatedin the same way as for the mixture of methyl esters of Example 2, usinghowever tetrahydro-2H-pyran-4-ol and sodium tetrahydro-2H-pyran-4-ylateinstead of methyl alcohol and sodium methylate and heating on a waterbath, and using 5 g of internal ester mixture and 727.5 mg (5.86 mM) ofsodium tetrahydro-2H-pyran-4-ylate. Washing of the Dowex resin iscarried out with tetrahydro-2H-pyran-4-yl alcohol and the residueobtained by evaporation of the filtered substance is dissolved in 50 mlof chloroform/ethanol 1:1. The yield in raw product is 5.3 g.Purification is also carried out as in Example 3. The yield in rawproduct is 5.3 g. Purification is also carried out as in Example 3. Theyield in purified ester mixture: 4.5 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel platesa freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.48-0.72 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 56 1-Heptyl ester of the ganglioside GM1

The 1-heptyl ester of GM₁ is prepared and isolated in the same manner asfor the methyl ester in Example 1, using however 1-heptanol and sodium1-heptylate in the place of methyl alcohol and sodium methylate andheating on a water bath, and using the same molar quantities of internalester and sodium 1-heptylate as in Example 1. Washing of the Dowex resinis effected with 1-heptyl alcohol and the residue obtained byevaporation of the filtered substance is dissolved in 50 ml of methylenechloride/1-heptanol 1:1. The yield in raw product is 3.9 g. Purificationis also carried out as in Example 1. Yield of the purified 1-heptylester of GM1 ganglioside: 4.3 g.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.89 and the absence of GM1 ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na₂ CO₃as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 57 Mixture of 1-heptyl esters of a mixture of gangliosides

The mixture of 1-heptyl esters is prepared and isolated in the same wayas for the mixture of methyl esters of Example 2, using however1-heptanol and sodium 1-heptylate instead of methyl alcohol and sodiummethylate and heating on a water bath, and using 5 g of internal estermixture and 809.8 mg (5.86 mM) of sodium 1-heptylate. Washing of theDowex resin is carried out with 1-heptyl alcohol and the residueobtained by evaporation of the filtered substance is dissolved in 50 mlof chloroform/ethanol 1:1. The yield in raw product is 5.2 g.Purification is also carried out as in Example 2. The yield in rawproduct is 5.2 g. Purification is also carried out as in Example 2. Theyield in purified ester mixture: 4.3 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.68-0.90 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 58 2-methyl-1-pentyl ester of the ganglioside GM₁

The 2-methyl-1-pentyl esteer of GM₁ is prepared and isolated in the samemanner as for the methyl ester in Example 1, using however2-methyl-1-pentanol and sodium 2-methyl-1-pentylate in the place ofmethyl alcohol and sodium methylate and heating on a water bath, andusing the same molar quantities of internal ester and sodium2-methyl-1-pentylate as in Example 1. Washing of the Dowex resin iseffected with 2-methyl-1-pentyl alcohol and the residue obtained byevaporation of the filtered substance is dissolved in 50 ml of methylenechloride/2-methyl-1-pentanol 1:1. The yield in raw product is 5.1 g.Purification is also carried out as in Example 1. Yield of the purified2-methyl-1-pentyl ester of GM₁ ganglioside: 4.4 g.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.90 and the absence of GM₁ ganglioside and itsinternal Na₂ CO₃ as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 59 Mixture of 2-methyl-1-pentyl esters of a mixture ofgangliosides

The mixture of 2-methyl-1-pentyl esters is prepared and isolated in thesame way as for the mixture of methyl esters of Example 2, using however2-methyl-1-pentanol and sodium 2-methyl-1-pentylate instead of methylalcohol and sodium methylate and heating on a water bath, and using 5 gof internal ester mixture and 727.7 mg (5.86 mM) and sodium2-methyl-1-pentylate. Washing of the Dowex resin is carried out with2-methyl-1pentyl alcohol and the residue obtained by evaporation of thefiltered substance is dissolved in 50 ml of chloroform/ethanol 1:1. Theyield in raw product is 5.3 g. Purification is also carried out as inExample 2. The yield in purified ester mixture: 4.4 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetraammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.70-0.93 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 60 3-methyl-2-pentyl ester of the ganglioside GM₁

The 3-methyl-2-pentyl ester of GM₁ is prepared and isolated in the samemanner as for the methyl ester in Example 1, using however3-methyl-2-pentanol and sodium 3-methyl-2-pentylate in the place ofmethyl alcohol and sodium methylate and heating on a water bath, andusing the same molar quantities of internal ester and sodium3-methyl-2-pentylate as in Example 1. Washing of the Dowex resin iseffected with 3-methyl-2-pentyl alcohol and the residue obtained byevaporation of the filtered substance is dissolved in 50 ml of methylenechloride/3-methyl-2-pentanol 1:1. The yield in raw product is 5.1 g.Purification is also carried out as in Example 1. Yield of the purified3-methyl-2-pentyl ester of GM₁ ganglioside: 4.2 g.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.92 and the absence of GM₁ ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na₂ CO₃as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 61 Mixture of 3-methyl-2-pentyl esters of a mixture ofgangliosides

The mixture of 3-methyl-2-pentyl esters is prepared and isolated in thesame way as for the mixture of methyl esters of Example 2, using however3-methyl-2-pentanol and and sodium 3-methyl-2-pentylate instead ofmethyl alcohol and sodium methylate and heating on a water bath, andusing 5 g of internal ester mixture and 727.7 mg (5.86 mM) of sodium3-methyl-b 2-pentylate. Washing of the Dowex resin is carried out with3-methyl-2-pentyl alcohol and the residue obtained by evaporation of thefiltered substance is dissolved in 50 ml of chloroform/ethanol 1:1. Theyield in raw product is 5.3 g. Purification is also carried out as inExample 2. The yield in purified ester mixture: 4.4 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.72-0.95 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 62 3-methoxyethyl ester of the ganglioside GM₁

The 2-methoxyethyl ester of GM₁ is prepared and isolated in the samemanner as for the methyl ester in Example 1, using however2-methoxyethanol and sodium 2-methoxyethylate in the place of methylalcohol and sodium methylate and heating on a water bath, and using thesame molar quantities of internal ester and sodium 2-methoxyethylate asin Example 1. Washing of the Dowex resin is effected with 2-methoxyethylalcohol and the residue obtained by evaporation of the filteredsubstance is dissolved in 50 ml of methylene chloride/2-methoxyethanol1:1. The yield in raw product is 4.9 g. Purification is also carried outas in Example 1. Yield of the purified 2-methoxyethyl ester of GM₁ganglioside: 4.3.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.77 and the absence of GM₁ ganglioside and itsinternal ester (Rf 065 and 0.75, respectively). Hydrolysis with Na₂ CO₃as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 63 Mixture of 2-methoxyethyl esters of a mixture of gangliosides

The mixture of 2-methoxyethyl esters is prepared and isolated in thesame way as for the mixture of methyl esters of Example 2, using however2-methoxyethanol and sodium 2-methoxyethylate instead of methyl alcoholand sodium methylate and heating on a water bath, and using 5 g ofinternal ester mixture and 574.9 mg (5.86 mM) of sodium2-methoxyethylate. Washing of the Dowex resin is carried out with2-methoxyethyl alcohol and the residue obtained by evaporation of thefiltered substance is dissolved in 50 ml of chloroform/ethanol 1:1. Theyield in raw product is 5.2 g. Purification is also carried out as inExample 3. The yield in purified ester mixture: 4.3 g. IR spectroscopycarried out on KBr pellets, shows the typical band of the ester at 1750cm⁻¹. When it is chromatographed on silica gel plates with a freshlyprepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.51-0.78 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 64 1-methoxy-2-propyl ester of the ganglioside GM₁

The 1-methoxy-2-propyl ester of GM₁ is prepared and isolated in the samemanner as for the methyl ester in Example 1, using however1-methoxy-2-propanol and sodium 1-methoxy-2-propylate in the place ofmethyl alcohol and sodium methylate and heating on a water bath, andusing the same molar quantities of internal ester and sodium1-methoxy-2-propylate as in Example 1. Washing the Dowex resin iseffected with 1-methoxy-2-propyl alcohol and the residue obtained byevaporation of the filtered substance is dissolved in 50 ml of methylenechloride/1-methoxy-2-propanol 1:1. The yield in raw product is 4.9 g.Purification is also carried out as in Example 1. Yield of the purified1-methoxy-2-propyl ester of GM₁ ganglioside: 4.2 g.

IR spectroscopic examination on KBr pellets shows the typical band ofthe ester at 1750 cm⁻¹. Chromatographic analysis of the product carriedout in the same way as in Example 1 shows the presence of a unitarycompound with an Rf of 0.81 and the absence of GM₁ ganglioside and itsinternal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na₂ CO₃as described in Example 1 produces the ganglioside GM₁.

EXAMPLE 65 Mixture of 1-methoxy-2-propyl esters of a mixture ofgangliosides

The mixture of 1-methoxy-2-propyl esters is prepared and isolated in thesame way as for the mixture of methyl esters of Example 2, using however1-methoxy-2-propanol and sodium 1-methoxy-2-propylate instead of methylalcohol and sodium methylate and heating on a water bath, and using 5 gof internal ester mixture and 657.0 mg (5.86 mM) of sodium1-methoxy-2-propylate. Washing of the Dowex resin is carried out with1-methoxy-2-propyl alcohol and the residue obtained by evaporation ofthe filtered substance is dissolved in 50 ml of chloroform/ethanol 1:1.The yield in raw product is 5.2 g. Purification is also carried out asin Example 2. The yield in purified ester mixture: 4.3 g.

IR spectroscopy, carried out on KBr pellets, shows the typical band ofthe ester at 1750 cm⁻¹. When it is chromatographed on silica gel plateswith a freshly prepared solution of chloroform/methanol/hydroxide oftetramethylammonium 1M 55:45:10 and determined with Ehrlich reagent, theproduct shows an Rf of 0.52-0.80 (ganglioside mixture 0.20-0.60).Complete transesterification is demonstrated in the same way asdescribed in Example 2.

EXAMPLE 66 Cyclohexyl ester of the ganglioside GM₁

5 g. of potassium salt (3.4 mM) of the ganglioside GM₁ are dissolved in50 ml of DMSO and 661.5 mg (3.75 mM) of bromocyclohexane and 625 mg(3.75 mM) of KI are added to the solution. It is left to react for 48hours at 25° C. At the end of the reaction the solution is partitionedwith n-butanol/H₂ O 2:1 to eliminate the DMSO and the salts. The butanolsolution is evaporated by drying and the residue is gathered in 50 ml ofchloroform/methanol 1:1 and the product is precipitated with 250 ml ofacetone. The raw product thus obtained (5.2 g) is then purified bypreparative column chromatography on silica gel using as solvent amixture of chloroform/methanol/water 60:35:8. The pure fractions aremixed, evaporated, redissolved in 15 ml of chloroform/isopropanol 1:1and the product is precipitated with 75 ml of acetone. Pure cyclohexylester yield 4.4 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterbond at 1750 cm⁻¹. Chromatographed on silica gel plates withchloroform/methanol/CaCl₂ at 0.3% 60:40:9 and withchloroform/methanol/ammonia 2.5 N 55:45:10 and determined with Ehrlichreagent, the product proves to be a uknitary compound with Rf of 0.70and 0.58 respectively and to be free from starting ganglioside GM₁ (Rf0.40 and 0.45 respectively). Treatment with a solution of Na₂ CO₃ 0.1 Nat 60° for an hour causes the splitting of the ester bond, givingstarting ganglioside GM₁.

EXAMPLE 67 Undecyl ester of the ganglioside GM₁

5 g. of potassium salt (3.14 mM) of the ganglioside GM₁ are dissolved in50 ml of DMSO and 882.1 mg (3.75 mM) of 1-bromoundecane and 625 mg (3.75mM) of KI are added to the solution. It is left to react for 48 hours at25° C. At the end of the reaction the solution is partitioned withn-butanol/H₂ O 2:1 to eliminate the DMSO and the salts. The butanolsolution is evaporated by drying and the residue is gathered in 50 ml ofchloroform/methanol 1:1 and the product is precipitated with 250 ml ofacetone. The raw product thus obtained (5.3 g) is then purified bypreparative column chromatography on silica gel using as solvent amixture of chloroform/methanol/water 60:35:8. The pure fractions aremixed, evaporated, redissolved in 15 ml of chloroform/isopropanol 1:1and the product is precipitated with 75 ml of acetone. Pure undecylester yield 4.9 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterbond at 1750 cm⁻¹. Chromatographed on silica gel plates withchloroform/methanol/CaCl₂ at 0.3% 60:40:9 and withchloroform/methanol/ammonia 2.5 N 55:45:10 and determined with Ehrlichreagent, the product proves to be a unitary compound with Rf of 0.68 and0.56 respectively and to be free from starting ganglioside GM₁ (Rf 0.40and 0.45 respectively). Treatment with a solution of Na₂ CO₃ 0.1 N at60° for an hour causes the splitting of the ester bond, giving startingganglioside GM₁.

EXAMPLE 68 1-hydroxy-undecan-11-yl ester of the ganglioside GM₁

5 g of potassium salt (3.14 mM) of the ganglioside GM₁ are dissolved in50 ml of DMSO and 0.42 mg (3.75 mM) of 11-bromo-1-undecanol and 625 mg(3.75 mM) of KI are added to the solution. It is left to react for 48hours at 25° C. At the end of the reaction the solution is partitionedwith n-butanol/H₂ O 2:1 to eliminate the DMSO and the salts. The butanolsolution is evaporated by drying and the residue is gathered in 50 ml ofchloroform/methanol 1:1 and the product is precipitated with 250 ml ofacetone. The raw product thus obtained (5.3 g) is then purified bypreparative column chromatography on silica gel using as solvent amixture of chloroform/methanol/water 60:35:8. The pure fractions aremixed, evaporated, redissolved in 15 ml of chloroform, isopropanol 1:1and the product is precipitated with 75 ml of acetone. Pure11-hydroxy-undecan-11-yl ester yield 4.8 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterbond at 1750 cm⁻¹. Chromatographed on silica gel plates withchloroform/methanol/CaCl₂ at 0.3% 60:40:9 and withchloroform/methanol/ammonia 2.5 N 55:45:10 and determined with Ehrlichreagent, the product proves to be a unitary compound with Rf of 0.65 and0.52 respectively and to be free from starting ganglioside GM₁ (Rf 0.40and 0.45 respectively). Treatment with a solution of Na₂ CO₃ 0.1 N at60° C. for an hour causes the splitting of the ester bond, giving theoriginal ganglioside GM₁.

EXAMPLE 69 Byanobutyr-4-yl ester of the ganglioside GM₁

5 g of potassium salt (3.14 mM) of the ganglioside GM₁ are dissolved in50 ml of DMSO and 550 mg (3.75 mM) of 4-bromobutyronitrile and 625 mg(3.75 mM) of KI are added to the solution. It is left to react for 48hours at 25° C. At the end of the reaction the solution is partitionedwith n-butanol/H₂ O 2:1 to eliminate the DMSO and the salts. The butanolsolution is evaporated by drying and the residue is gathered in 50 ml ofchloroform/methanol 1:1 and the product is precipitated with 250 ml ofacetone. The raw product thus obtained (5.1 g) is then purified bypreparative column chromatography on silica gel, using as solvent amixture of chloroform/methanol/water 60:35:5.

The pure fractions are mixed, evaporated, redissolved in 15 ml ofchloroform/isopropanol 1:1 and the product is precipitated with 75 ml ofacetone. Pure cyanobutyr-4-yl ester yield 4.6 g.

IR spectroscopy, carried out on KBr pellets, shows the typical esterbond at 1750 cm⁻¹. Chromatographed on silica gel plates withchloroform/methanol/CaCl₂ at 0.3% 60:40:9 and withchloroform/methanol/ammonio 2.5 N 55:45:10 and determined with Ehrlichreagent, the product proves to be a unitary compound with Rf of 0.42 and0.45 respectively and to be free starting ganglioside GM₁ (Rf 0.40 and0.45 respectively). Treatment with a solution of Na₂ CO₃ 0.1 N at 60° C.for an hours causes the splitting of the ester bond, giving startingganglioside GM₁.

EXAMPLE 70 Pyrrolidine amide of the ganglioside GM₁

This derivative is prepared from 5 g of internal ester of theganglioside GM₁ (3.27 mM) and from 25 ml of pyrrolidine in the same wayas in Example 16 and the same purification method is also followed. Ayield of 5.1 g of pure pyrrolidine amide of the ganglioside GM₁ isobtained.

The IR spectroscopic data are the same as for the methylamide in Example16 and chromatographic examination in the same conditions as in thatExample proved the product to be unitary and free from GM₁ with Rf of0.29 and 0.46 respectively (Rf of GM₁, 0.35 and 0.40 respectively).

EXAMPLE 71 Mixture of pyrrolidine amides of a ganglioside mixture

This derivative mixture is prepared with 5 g of the mixture of internalesters of gangliosides used in Example 17 and 25 ml of pyrrolidine inthe same way as in Example 17, the same purification method is alsoused. A yield of 4.9 of the mixture of pyrrolidine amides of aganglioside mixture is obtained.

The Rf values determined by chromatography on silica gel plates withchloroform/methanol/ammonia 4 N 55:45:10 and chloroform/methanol/CaCl₂at 0.3% 60:35:8 and determined with resorcinol reagent proved to be0.08-0.40 and 0.35-0.48 respectively (Rf of the original mixture ofgangliosides 0.15-0.70 and 0.05-0.40 respectively).

EXAMPLE 72 Piperidine amide of the ganglioside GM₁

This derivative is prepared from 5 g of internal ester of theganglioside GM₁ (3.27 mM) and from 25 ml of piperidinie in the same wayas in Example 16 and the same purification method is also followed. Ayield of 5.2 g of pure piperidine amide of the ganglioside GM₁ isobtained.

The IR spectroscopic data are the same as for the methylamide in Example16 and chromatographic examination in the same conditions as in thatExample proved the product to be unitary and free from GM₁ with Rf of0.30 and 0.50 respectively (Rf of GM₁, 0.35 and 0.40 respectively).

EXAMPLE 73 Mixture of piperidine amides of a ganglioside mixture

This derivative mixture is prepared with 5 g of the mixture of internalesters of gangliosides used in Example 17 and 25 ml of piperidine in thesame way as in Example 17, the same purification method is also used. Ayield of 5.1 g of the mixture of piperidine amides of a gangliosidemixture is obtained.

The Rf values determined by chromatography on silica gel plates withchloroform/methanol/ammonia 4 N 55:45:10 and chloroform/methanol/CaCl₂at 0.3% 60:35:8 and determined with resorcinol reagent proved to be0.10-0.42 and 0.37-0.50 respectively (Rf of the original mixture ofgangliosides 0.15-0.70 and 0.05-0.40 respectively).

EXAMPLE 74 Tetrahydrofurfuryl amide of the ganglioside GM₁

This derivative is prepared from 5 g of internal ester of theganglioside GM₁ (3.27 mM) and from 25 Ml of tetrahydrofurfurylamine inthe same way as in Example 16 and the same purification method is alsofollowed. A yield of 5.3 g of pure tetrahydrofurfuryl amide of theganglioside GM₁ is obtained.

The IR spectroscopic data are the same as for the methylamide in Example16 and chromatographic examination in the same conditions as in theExample proved the product to be unitary and free from GM₁ with Rf of0.33 and 0.52 respectively (Rf of GM₁, 0.35 and 0.40 respectively).

EXAMPLE 75 Mixture of tetrahydrofurfuryl amides of a ganglioside mixture

This derivative mixture is prepared with 5 g of the mixture of internalesters of gangliosides used in Example 17 and 25 ml oftetrahydrofurfurylamine in the same way as in Example 17, the samepurification method is also used. A yield of 5.2 g of the mixture oftetrahydrofurfuryl amides of a ganglioside mixture is obtained.

The Rf values determined by chromatography on silica gel plates withchloroform/methanol/ammonia 4 N 55:45:10 and chloroform/methanol/CaCl₂at 0.3% 60:35:8 and determined with resorcinol reagent proved to be0.12-0.45 and 0.40-0.57 respectively (Rf of the original mixture ofgangliosides 0.15-0.70 and 0.05-0.40 respectively).

EXAMPLE 76 2-Methylpiperidine amide of the ganglioside GM₁

This derivative is prepared from 5 g of internal ester of theganglioside GM₁ (3.27 mM) and from 25 ml of 2-methyl-piperidine amine inthe same way as in Example 16 and the same purification method is alsofollowed. A yield of 5.2 g of pure 2-methylpiperidine amide of theganglioside GM₁ is obtained.

The IR spectroscopic data are the same as for the methylamide in Example16 and chromatographic examination in the same conditions as in thatExample proved the product to be unitary and free from GM₁ with Rf of0.33 and 0.54 respectively (Rf of GM₁, 0.35 and 0.40 respectively).

EXAMPLE 77 Mixture of 2-methylpiperidine amides of a ganglioside mixture

This derivative mixture is prepared with 5 g of the mixture of internalesters of gangliosides used in Example 17 and 25 ml of2-methylpiperidine in the same as in Example 17 and 25 of2-methylpiperidine in the same way as in Example 17, the samepurification method is also used. A yield of 5.4 g of the mixture of2-methylpiperidine amides of a ganglioside mixture is obtained.

The Rf values determined by chromatography on silica gel plates withchloroform/methanol/ammonia 4 N 55:45:10 and chloroform/methanol/CaCl₂at 0.3% 60:35:8 and determined with resorcinol reagent proved to be0.14-0.46 and 0.39-0.59 respectively (Rf of the original mixture ofgangliosides 0.15-0.70 and 0.05-0.40 respectively).

EXAMPLE 78 1-Methyl-piperazine amide of the ganglioside GM₁

This derivative is prepared from 5 g of internal ester of theganglioside GM₁ (3.27 mM) and from 25 ml of 1-methylpiperazine in thesame way as in Example 16 and the same purification method is alsofollowed. A yield of 5.1 g of pure 1-methyl-piperazine amide of theganglioside GM₁ is obtained. The IR spectroscopic data are the same asfor the methylamide in Example 16 and chromatographic examination in thesame conditions as in that Example proved the product to be unitary andfree from GH₁ with Rf of 0.04 and 0.08 respectively (Rf of GM₁, 0.35 and0.40 respectively).

EXAMPLE 79 Mixture of 1-methyl-piperazine amides of a gangliosidemixture

This derivative mixture is prepared with 5 g of the mixture of internalesters of gangliosides used in Examples 17 and 25 ml of1-methyl-piperazine in the same way as in Example 17, the samepurification method is also used. A yield of 5.5 of the mixture of1-methyl-piperazine amides of a ganglioside mixture is obtained.

The Rf values determined by chromatography on silica gel plates withchloroform/methanol/CaCl₂ at 0.3% 60:35:8 and determined with resorcinolreagent to be 0.01-0.06 and 0.01-0.12 respectively (Rf of the originalmixture of gangliosides 0.15-0.70 and 0.05-0.40 respectively).

EXAMPLE 80 2-Phenylethyl amide of the ganglioside GM₁

This derivative is prepared from 5 g of internal ester of theganglioside GM₁ (3.27 mM) and from 25 ml of 2-phenyl-ethyl amine in thesame way as in Example 16 and the same purification method is alsofollowed. A yield of 5.3 g of pure 2-phenylethyl amide of theganglioside GM₁ is obtained.

The IR spectroscopic data are the same as for the methylamide in Example16 and chromatographic examination in the same conditions as in thatExample proved the product to be unitary and free from GM₁ with Rf of0.33 and 0.73 respectively (Rf of GM₁, 0.35 and 0.40 respectively).

EXAMPLE 81 Mixture of 2-phenylethyl amides of a ganglioside mixture

This derivative mixture is prepared with 5 g of the mixture of internalesters of gangliosides used in Example 17 and 25 ml of 2-phenylethylamine in the same way as in Example 17, the same purification method isalso used. A yield of 5.5 g of the mixture of 2-phenylethyl amides of aganglioside mixture is obtained.

The Rf values determined by chromatography on silica gel plates withchloroform/methanol/ammonia 4 N 55:45:10 and chloroform/methanol/CaCl₂at 0.3% 60:35:8 and determined with resorcinol reagent proved to be0.12-0.44 and 0.60-0.78 respectively (Rf of the original mixture ofgangliosides 0.15-0.70 and 0.05-0.40 respectively).

Pharmaceutical Preparations

As discussed above, one of the objects of the present invention residesin pharmaceutical preparations containing, as an active substance, oneor more of the new ganglioside derivatives described above and, inparticular, those derived from ganglioside groups A and B, as well asthe specific ones listed previously. Furthermore, the pharmaceuticalpreparations of the present invention include esterified derivatives oramides and their acylated derivatives, including those already known andthose novel derivatives described above. Particularly preferred are themethyl esters of gangliosides G_(M1) and G_(M3) and their peracylatedderivatives, as well as the amide of ganglioside G₃.

The pharmaceutical preparations of the invention can be preparations fororal, rectal, parenteral, local or intradermal use. They are thereforein solid or semisolid form, such as pills, tablets, gelatine coveredcapsules, capsules, suppositories or soft gelatine capsules. Forparenteral use, it is possible to use those forms in intended forintramuscular, subcutaneous or intradermal administration or intendedfor infusions or intravenous injections, and which may, therefore, bepresented as solutions of the active compounds or as freeze-driedpowders of the active compounds to be added to one or morepharmaceutically acceptable excipients or diluents, suitable for theabove uses and having osmolarity which is compatible with physiologicalliquids. Preparations in the form of sprays, such as nasal sprays,creams or ointments for topical use of suitably prepared plasters forintradermal administration can be utilized for local application. Thepreparations of the invention can be administered to humans or animals.Preferably, the compositions should contain between 0.01% and 10% of theactive component, for solutions, sprays ointments and creams and between1% and 100% and preferably between 5% and 50% of the active compound forthe solid preparations. The dosage to be administered depends on medicalindication, on the desired effect and on the chosen administrationroute. Examples 82-83 illustrated the pharmaceutical preparationsaccording to the invention.

EXAMPLE 82 Pharmaceutical preparations in solution for injectionPreparation No. 1

one 2 ml vial contains:

active substance: mg 5

sodium chloride: mg 16

citrate buffer pH 6 in water distilled pyrogen free: q.b.a. ml 2

The active substance may be chosen from the group composed of theganglioside derivatives described in any of Examples: 14, 15, 16, 17,18, 19, 20, 21, 22, 23 and 24.

Preparation No. 2 one 2 ml vial contains:

active substance: mg 50

sodium chloride: mg 16

citrate buffer pH 6 in water distilled pyrogen free: q.b.a. ml 2

The active substance may be chosen from the group composed of theganglioside derivatives described in any of Examples: 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and 39.

Preparation No. 3

one 4 ml flacon contains:

active substance: mg 100

sodium chloride: mg 32

citrate buffer pH 6 in water distilled pyrogen free: q.b.a. ml 4

The active substance may be chosen from the group composed of theganglioside derivatives described in any of Examples: 20-39.

Preparations Nos. 1, 2 and 3 may be administered directly to animals orhumans by one of the routes describes above. The compounds may alsocontain other active substance.

EXAMPLE 83 Pharmaceutical compositions prepared in double flacons

The preparations illustrated in this Example are obtained with doubleflacons. The first flacon contains the active substance in the form of afreeze-dried powder in quantities which may vary between 10% and 90% inweight, together with a pharmaceutically acceptable excipient, withglycine or mannitol. The second flacon contains the solvent, such as asolution of sodium chloride and a citrate buffer.

The contents of the two flacons are mixed immediately before use and thefreeze-dried powder of the active substance dissolves rapidly, giving aninjectable solution. Flacons containing freeze-dried powder of theactive substance are the preferable pharmaceutical form of the presentinvention.

System No. 1

a. one freeze-dried 2 ml flacon contains:

active substance: mg 5

glycine: mg 30 b.

one 2 ml vial of solvent contains:

sodium chloride: mg 16

citrate buffer in pyrogen free water: q.b.a.ml 2

The active substance may be chosen from the group composed of theganglioside derivatives described in any of Examples: 14, 15, 16, 17,18, 19, 20, 21, 22, 23 and 24.

System No. 2

a. one 3 ml freeze-dried vial contains:

active substance: mg 5

mannitol: mg 40 b.

one 2 ml vial of solvent contains:

sodium chloride: mg 16

citrate buffer in pyrogen free water: q.b.a.ml 2

The active substance may be chosen from the group composed of theganglioside derivatives described in any of Examples: 1, 2, 4, 5, 6, 7,8, 9, 10, 11, 12 and 13.

System No. 3

a. one freeze-dried 3 ml vial contains:

active substance: mg 50

glycine: mg 25

b. one 3 ml vial of solvent contains:

sodium chloride: mg 24

citrate buffer in pyrogen free water: q.b.a.ml 3

The active substance may be chosen from the group of gangliosidederivatives described in any of Examples 14, 15, 16, 17, 21, 37, 38 and39.

System No. 4

a. one freeze-dried 3 ml vial contains:

active substance: mg 50

mannitol: mg 20

b. one 3 ml vial of solvent contains:

sodium chloride: mg 24

citrate buffer in pyrogen free water: q.b.a. ml 3

The active substance may be chosen from the group of gangliosidederivatives described in any of Examples 10-13 and 66-67.

System No. 5

a. one freeze-dried 5 ml vial contains:

active substance: mg 150

glycine: mg 50

b. one 4 ml vial of solvent contains:

sodium chloride: mg 32

citrate buffer in pyrogen free water: q.b.a. ml 4

The active substance may be chosen from the group composed of theganglioside derivatives described in any of Examples 14-39.

System No. 6

a. one freeze-dried 5 ml flacon contains:

active substance: mg 100

mannitol: mg 40

b. one 4 ml vial of solvent contains:

sodium chloride: mg 32

citrate buffer in pyrogen free water: q.b.a. ml 4

The active substance may be chosen from the group of gangliosidederivatives described in any of Examples 5-9.

System No. 7

a. one 3 ml flacon contains:

sterile micronized active substance: mg 40

b. one 3 ml vial of solvent contains:

Tween 80: mg 10

sodium chloride: mg 24

phosphate buffer in pyrogen free water: q.b.a. ml 3

The active substance may be chosen from the group of gangliosidederivatives described in any of Examples 40-45.

System No. 8

a. one 5 ml flacon contains:

sterile micronized active substance: mg 100

b. one 4 ml vial of solvent contains:

Tween 80: mg 15

soya lecithin: mg 5

sodium chloride: mg 36

citrate buffer in pyrogen free water: q.b.a. ml 4

The active substance may be chosen from the group of gangliosidederivatives described in any of Examples 40-45.

Therapeutic Activity

All of the new ganglioside derivatives discussed above, and inparticular those gangliosides of groups A and B and the above-listedspecific compounds, comprise the active ingredients of thepharmaceutical preparations of the present invention. Furthermore, thesepharmaceutical preparations may also contain some gangliosidederivatives of the type described above and already known in literature,such as the methyl ester of ganglioside G_(M1) or its peracetylatedderivative. The invention also includes the application of all theseganglioside derivatives, both new and already known, for therapeuticuse.

As discussed above, the therapeutic action of gangliosides and somederivatives, such as those of the present invention, is due to astimulation of the sprouting phenomena of the nervous cell, due to whichit is possible to obtain a recovery of nervous conduction. For example,the in vivo administration of a ganglioside mixture obtained from bovinebrain as described in Example 2 (GA mixture) provokes sprouting of thesciatic nerve in rats after crushing, and can, therefore, aid inrecovery of electric activity of the nerve at the neuromuscular jointlevel.

Since neurite sprouting may be considered as a localized neuronaldifferentiation, the biochemical mechanisms by which gangliosidemolecules produce this effect were studied on the basis of their effecton cellular differentiation, using cellular cultures, in vitro, ofpheocromocytoma PC₁₂. The addition of gangliosides to nerve growthfactor (NGF), which is an inducer of differentiation of PC₁₂ cells, inthe PC₁₂ culture medium stimulates neuronal sprouting. This effect maybe attributed to the incorporation of gangliosides in the neuronalmembrane which induces a modification of its functional properties, thatis to say, the enzymatic activities. Indeed, the incorporation ofgangliosides in the neuronal membrane is able to stimulate activity of(Na⁺, K⁺)-ATPase. To emphasize the importance of the activation of thisenzyme connected to the membrane, it should be remembered that someauthors trace the survival and consequently the differentiation ofneuronal cells in culture back to an activation of this enzyme by NGF.On the other hand, the key role played by this enzyme in electricactivity is well known, as it is involved in the ionic mechanismsubjected to the propagation of nervous impulses along the axonalmembrane.

On the basis of the methods described above, determinations were made ofthe pharmacological activities expressed by neurite sprouting in thePC₁₂ cells for the new ganglioside derivatives according to the presentinvention. Results obtained with (Na⁺, K⁺) ATPase in vitro of somederivatives are also reported.

Effects of ganglioside derivatives on neurite sprouting in PC12 cells

Materials and methods:

The PC₁₂ cell line (derived from a subclone 1A) was supplied by Dr. P.Calissano (C.N.R. Laboratory of Cellular Biology--Rome).

The cells (100,000/plate) are kept at 37° C. in a Heraeus incubator (5%CO₂ and 95% humidified air) and resown on 60 mm Falcon Integrid plateson a collagene support in the presence of the following culture medium:85% RPM 1640 (Gibco), 10% heat-inactivated equine serum, 5% fetal calfserum (Gibco), 50 U/ml penicillin and 25 mg/ml of streptomycine. Thecells are then washed three times in a serum-free vehicle. After threewashes, the cells are incubated in a serum-free vehicle with 50 ng/ml ofNGF and with the ganglioside derivative according to the invention orwith the ganglioside mixture used as a comparison at concentrations of10⁻⁶ M. The addition of the serum-free vehicle with NGF (50 ng/ml) hasthe effect of interrupting the proliferation of the cells, formingneurites and obtaining the differentiation as early as the third day.This effect is evaluated by counting the number of cells with neuritesat the third day.

Results

The results are reported in the following Table 1 using as comparisonvalues those obtained with ganglioside mixture obtained according to theprocedure described in Example 2 and the single monosialogangliosidefraction G_(M1).

                  TABLE 1                                                         ______________________________________                                        Effects of gangliosides and their derivatives according                       to the present invention, on neurite sprouting in PC.sub.12 cells.                                          % of the no.                                                                  of cells with                                                        Concen-  neurites at                                     Compound             tration  3rd day                                         ______________________________________                                        Controls             10.sup.-6 M                                                                            21.5                                            Ganglioside mixture GA        39.5                                            (see Example 2)                                                               Monosialoganglioside G.sub.Ml                                                                      "        34.8                                            Methylic ester of GA "        32.8                                            Methylic ester of G.sub.Ml                                                                         "        35.7                                            Ethylic ester of GA  "        34.9                                            Ethylic ester of G.sub.Ml                                                                          "        37.1                                            Isopropylic ester of GA                                                                            "        37.3                                            Isopropylic ester of G.sub.Ml                                                                      "        37.0                                            Tertiarbutylic ester of GA                                                                         "        29.5                                            Tertiarbutylic ester of G.sub.Ml                                                                   "        32.3                                            Benzylic ester of GA "        31.4                                            Benzylic ester of G.sub.Ml                                                                         "        28.3                                            Allylic ester of GA  "        34.1                                            Allylic ester of G.sub.Ml                                                                          "        31.5                                            Etoxycarbonylmethylic ester of G.sub.Ml                                                            "        27.3                                            Amide of GA          "        36.3                                            Amide of G.sub.Ml    "        33.3                                            Methylamide of GA    "        40.0                                            Ethylamide of GA     "        35.0                                            Ethylamide of G.sub.Ml                                                                             "        32.2                                            Benzylamide of GA    "        26.7                                            Benzylamide of G.sub.Ml                                                                            "        30.8                                            Isopropylamide of G.sub.Ml                                                                         "        31.4                                            Dimethylamide of GA  "        29.3                                            Dimethylamide of G.sub.Ml                                                                          "        34.3                                            Diethylamide of GA   "        25.0                                            Diethylamide of G.sub.Ml                                                                           "        28.5                                            Ethylmethylamide of G.sub.Ml                                                                       "        35.3                                            3-dimethylaminopropyl-1-amide of GA                                                                "        32.9                                            3-dimethylaminopropyl-1-amide of G.sub.Ml                                                          "        37.3                                            Dimethylaminoethylamide of G.sub.Ml                                                                "        34.8                                            Ethanolamide of GA   "        38.3                                            Ethanolamide of G.sub.Ml                                                                           "        41.2                                            6-hydroxyhexyl-1-amide of G.sub.Ml                                                                 "        36.4                                            Peracetylated GA mixture                                                                           "        28.5                                            Peracetylated G.sub.Ml                                                                             "        30.2                                            Methylic ester of peracetylated GA                                                                 "        33.6                                            Methylic ester of peracetylated G.sub.Ml                                                           "        29.4                                            Amide of peracetylated GA                                                                          "        25.3                                            Amide of peracetylated G.sub.Ml                                                                    "        31.4                                            ______________________________________                                    

Effects of ganglioside derivatives on (Na+, K+) ATPase of the neuronalmembrane

Materials and methods:

a. Preparation of the raw mitochondrial fraction of rat brain (FractionP2):

The procedure used in the preparation of P₂ fraction was taken fromMorgan e coll., Biochem. Biophys. Acta 241, 37 (1971). (The variousoperations were carried out at 0° C. to 4° C.; the xg values are averagecentrifugal forces).

Adult male Sprague Dawley rats (supplied by Charles River) (body weight150-175 g) were decapitated and their brains were immediately removedand washed in an ice cold isotonic solution. After excision of thecerebellum, the brains were homogenized by effecting 12 up and downmovements in a motor-driven glass and Teflon homogenzier (declaredradial clearance 0.25 mm; 800 r.p.m.) using 4 vol. of homogenizingsolution (0.32M saccharose containing 1 mM potassium phosphate bufferand 0.1 nM of disodium EDTA, pH 7.27). The homogenized substance,filtered through four layers of fine gauze, was centrifuged at 1000r.p.m. for 15 minutes. The resulting pellet was washed with the sameinitial volume of homogenizing solution and centrifuged as describedabove. The gathered supernatants were centrifuged at 17,500 r.p.m. for25 minutes (these conditions of gravitational centrifugal force wereused to obtain the maximum enrichment of nervous termination in thefraction) and the pellet was washed four times in 9 volumes (each time)of homogenizing solution and centrifuged (17,500 r.p.m. for 25 minutes).

The final pellet, known as "P₂ fraction" contains as its principalcomponent, whole mitochondria and nervous terminations. The final pelletwas homogeneously resuspended with a suitable volume of homogenizingsolution with a glass and Teflon homogenizer, and used immediately forthe test. In order to avoid inconsistencies due to conservation of thematerial, fresh P₂ fractions were prepared before each use. Thepreparations of P₂ fractions has a ganglioside content of bound 33.9±2.8(S.D.) n moles of sialic acid per mg of protein.

b. Activation of the ATPase enzyme:

ATPase activity was measured by spectrophotometry according to Wallickand coll. [J. Pharm. Exptl. Therap. 189, 434, (1979)]. The reactionmixture, unless otherwise indicated, is composed of: 50 mM ofsaccharose, 0.2 mM of disodium EDTA (brought to pH 7.4), 100 mM NaCl, 5mM MgCl₂, 10 mM KCl, 2 mM of monopotassic salt of phospho(enol)pyruvate(PEP) (brought to pH 7.4), 3 mM ATP, 50 mM TRIS-HCl pH 7.4, 0.33 mMNADH, pyruvate-kinase (PK) (30 g/ml) and lactate dehydrogenase (LDH) 10g/ml) in a final volume of 3 ml and with a final pH of 7.2. The reactionis begun with the addition of 50-75 μg (as a protein) of the P₂fraction. The (Na⁺, K⁺)ATPase activity is determined by the differencebetween the total ATPase activity and the dependent ATPase Mg²⁺ activitymeasured in the presence of 3×10⁻⁵ M of Ouabain. The time taken for eachsingle test was 3-5 minutes. The ATPase activity was expressed asInternational Unit (I.U.) (μ moles hydrolized ATP/mg protein/min). Theactivity of the ganglioside derivatives (50 nM) was dosed by carryingout incubation with the neuronal membranes at 37° C. for two hours.

Results

The results of the comparative studies on ATPase activity are reportedin Table 2.

                  TABLE 2                                                         ______________________________________                                        Effects of gangliosides and their derivatives on the                          (Na.sup.+, K.sup.+)ATPase of the neuronal membrane                                                         % increase in                                                                 (Na.sup.+, K.sup.+)                              Product       Concentration  ATPase activity                                  ______________________________________                                        Controls      50             100                                              Ganglioside mixture GA                                                                      50             142                                              GA (see Example 2)                                                            G.sub.Ml      50             133                                              Methyl ester of GA                                                                          50             128                                              Amide of GA   50             139                                              Methyl ester of G.sub.Ml                                                                    50             132                                              Amide of G.sub.Ml                                                                           50             128                                              ______________________________________                                    

It should be noted that the ganglioside derivatives of the resentinvention have a more prolonged action in time compared to gangliosidesand are therefore useful as "retard" medicaments. This phenomenon isillustrated for example by the following experiments on the kinetics ofabsorption of ganglioside esters.

In vivo blood distribution of ganglioside derivatives

1. Preparation of labelled products

Labelled ganglioside is prepared as described by Suzuki et al. [J. LipidRes. 13, 687-690 (1972)] and modified by Ghidoni et al. [Ital. J.Biochem. 23, 320-328 (1974)]. The ethyl and isopropyl esters areprepared by esterification of this labelled ganglioside. The specificradioactivity is measured both of the ganglioside and esters.

2. Treatment of the animals

Swiss mice obtained from Charles River (Calco), weighing about 20-25 gwere treated by intravenous route with 10 μC of the products.2-4-8-12-16 hours after administration, the animals were sacrificed bydecapitation and their blood was gathered in heparinized flasks. Thenonvolatile radioactivity was determined on blood samples by means of aPackard TRICARB scintillator and then identified with reference to ³ Hganglioside and to the esters by TLC.

3. Results

After intravenous administration of tritiated ganglioside, the kineticcurve was biphasic, declining with a t/2 of about three hours followedby a slow elimination phase which remains constant until the sixteenthhour. With the ethyl and isopropyl ester of G_(M1) ganglioside, thelevels of product at first identified do not reach the maximum observedwith the natural product, but with time they reach higher maximums andremain at these levels for longer. In this way there is an increase inthe distribution volume and an increase in the time of maintenance ofthe therapeutic doses.

Due to the pharmacological properties described above, the gangliosidederivatives of the present invention may be used as drugs in varioustherapies for treatment of nervous system pathologies, in particulartherapies of the peripheral nerves and those of the central nervoussystem.

The invention particularly regards the therapeutic use of theganglioside derivatives which are specifically mentioned above, such asthe groups of derivatives of gangliosides A and B and those specified indetail and the administration of all these derivatives at the doseshereafter reported. More particularly, these ganglioside derivatives maybe used in therapy of peripheral nervous system pathologies oftraumatic, compressive, generative, or toxic-infective origin, in whichit is necessary to stimulate nervous regeneration and recovery ofneuromuscular function and in pathologies of the central nervous systemof traumatic, anoxic, degenerative or toxic-infective origin in which itis necessary to stimulate neuronal sprouting phenomena in order toobtain functional recovery. Due to their retard effect, the gangliosidederivatives of the present invention have a clear advantage overgangliosides themselves, which have until now been the drugs used in thecases reported above.

The dosages for administration will depend on the desired effect and onthe chosen administration route. Administration is usually effected byintramuscular, subcutaneous, intravenous, intradermal or pulmonal route,preferably in a suitably buffered aqueous vehicle. The pharmaceuticalform for conserving the substance may in this case be vials containingsolutions of the derivatives, possibly with other auxiliary ingredients,as described for the pharmaceutical preparations of the presentinvention. For therapeutic or possibly also prophylactic application bythe above mentioned parenteral route, the dosage may vary preferablybetween 0.05 mg and 5 mg of active substance per Kg of body weight/dieand especially between 0.05 mg and 2 mg per Kg of body weight/die.

Although the new therapeutic applications according to the invention aregenerally suitable for all pathologies connected with nervous stimulusconduction in the central and peripheral nervous systems, the followingspecific pathologies are of particular interest: retrobulbar opticalneurities, paralysis of the oculomotor nerves, neuritis of thetrigeminus, Bell's palsy and paralysis of the facial nerve, Garcin'ssyndrome, radiculitis, traumatic lesions of the peripheral nerves,diabetic and alcoholic polyneuritis, obstetric paralysis, paralyticsciatica, motoneuron diseases, myelopathic muscular amyotrophic-atrophiclateral sclerosis, progressive bulbar paralysis, severe myasthenia,Lambert Eaton syndrome, muscular dystrophy, deterioration of synapticnervous transmission in the CNS and PNS, and impairments inconsciousness such as confusional states, cerebral concusion,thrombosis, embolism.

We claim:
 1. A ganglioside derivative comprised of an oligosaccharidemoiety formed by 1 to 5 monosaccharides, at least one sialic acid moietyand at least one ceramide moiety, the carboxyl groups of said sialicacid moieties being esterified with an alcohol, said alcohol being asubstituted alcohol selected from the group consisting of a C₁₋₁₂aliphatic alcohol, an araliphatic alcohol having a maximum of 12 carbonatoms and with only one benzene ring, a heterocyclic alcohol with amaximum of 12 carbon atoms and with one heterocyclic ring containing ahetero atom selected from the group consisting of N, S and O, analicyclic alcohol with a maximum of 14 carbon atoms, and analiphaticalicyclic alcohol with a maximum of 14 carbon atoms andcontaining only one cycloaliphatic ring, wherein said substitutedalcohol is substituted with at least one member selected from the groupconsisting of hydroxyl, amine, C₁₋₄ alkoxyl, carboxyl, C₁₋₄ carbylcoxy,C₁₋₄ alkylamine and di-C₁₋₄ alkylamine or said substituted alcohol is anaraliphatic alcohol substituted with between 1 and 3C₁₋₄ alkyl groups.2. A ganglioside derivative according to claim 1, wherein saidsubstituted alcohol is an araliphatic alcohol with only one benzenering, and with a maximum of 4 carbon atoms in the aliphatic chain.
 3. Aganglioside derivative according to claim 2, wherein said C₁₋₄ alkylgroup is a methyl group.
 4. A ganglioside derivative according to claim1, wherein the basic gangliosides are selected from the group consistingof G_(M1), G_(D1a), G_(D1b) and G_(T1b).
 5. A ganglioside derivativeaccording to claim 2, wherein the basic gangliosides are selected fromthe group consisting of G_(M1), G_(D1a), G_(D1b) and G_(T1b).
 6. Aganglioside derivative according to claim 3, wherein the basicgangliosides are selected from the group consisting of G_(M1), G_(D1a),G_(D1b) and G_(T1b).
 7. A ganglioside derivative according to claim 1,wherein the hydroxyl groups of said ceramide moiety and saidoligosaccharide moiety are acylated with a carboxylic acid, saidcarboxylic acid being an aliphatic, aromatic, araliphatic, alicyclic orheterocyclic carboxylic acid with a maximum of 10 carbon atoms.
 8. Aganglioside derivative according to claim 4, wherein the hydroxyl groupsof said ceramide moiety and said oligosaccharide moiety are acylatedwith a carboxylic acid, said carboxylic acid being an aliphatic,aromatic, araliphatic, alicyclic or heterocyclic carboxylic acid with amaximum of 10 carbon atoms.
 9. A ganglioside derivative according toclaim 5, wherein the hydroxyl groups of said ceramide moiety and saidoligosaccharide moiety are acylated with a carboxylic acid, saidcarboxylic acid being an aliphatic, aromatic, araliphatic, alicyclic orheterocyclic carboxylic acid with a maximum of 10 carbon atoms.
 10. Aganglioside derivative according to claim 6, wherein the hydroxyl groupsof said ceramide moiety and said oligosaccharide moiety are acylatedwith a carboxylic acid, said carboxylic acid being an aliphatic,aromatic, araliphatic, alicyclic or heterocyclic carboxylic acid with amaximum of 10 carbon atoms.
 11. A ganglioside derivative according toclaim 1, selected from the group consisting of the peracetylates,perpropionylates, perbutyrrylates, maleinylates and succinylates of saidesters.
 12. A ganglioside derivative according to claim 4, selected fromthe group consisting of the peracetylates, perpropionylates,perbutyrrylates, maleinylates and succinylates of said esters.
 13. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent and, as an active ingredient, an effective nervepathology treatment amount of a ganglioside derivative according toclaim
 1. 14. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and, as an active ingredient, an effectivenerve pathology treatment amount of a ganglioside derivative accordingto claim
 7. 15. A method for the treatment of pathologies of the nervoussystem of one affected therewith, comprising administering thereto aneffective nerve pathology treatment amount of a ganglioside derivativeaccording to claim
 1. 16. A method for the treatment of pathologies ofthe nervous system of one affected therewith, comprising administeringthereto an effective nerve pathology treatment amount of a gangliosidederivative according to claim
 7. 17. A ganglioside derivative comprisedof an oligosaccharide moiety formed by 1 to 5 monosaccharides, at leastone sialic acid moiety and at least one ceramide moiety, the carboxylgroups of said sialic acid moieties being amidized with an amine, saidamine being selected from the group consisting of a C₁₋₆ alkylamine, asubstituted C₁₋₁₂ aliphatic amine, a substituted aromatic amine having amaximum of 12 carbon atoms, a substituted heterocyclic amine having amaximum of 12 carbon atoms and containing a heteroatom selected from N,S and O, a sustituted alicyclic amine having a maximum of 12 carbonatoms, and a substituted araliphatic amine with only one benzene ringand a maximum of 4 carbon atoms in the aliphatic portion, saidsubstituted amines being substituted with a group selected from thegroup consisting of amine, C₁₋₄ alkylamine, di-C₁₋₄ alkylamine,hydroxyl, C₁₋₄ alkoxyl, C₃₋₆ alkyleneamines and alkyl or alkylene groupswherein the carbon claims are interrupted with a N, O or S.
 18. Aganglioside derivative according to claim 17, wherein said C₁₋₆alkylamine is a member selected from the group consisting ofmethylamine, ethylamine, ethylmethylamine, propylamine, butylamine,hexylamine, dimethylamine, diethylamine, diisopropylamine, dihexylamineand benzylamine.
 19. A ganglioside derivative according to claim 17,wherein said substituted amine is a member selected from the groupconsisting of dimethylaminoethylamine, dimethylamino propyl-1- amine,6-hydroxyhexyl-1-amine, pyrrolidine, piperidine, azepine,ethylenediamine, trimethyldiamine, piperazine, aminoethanol,aminopropanol, morpholine and thiomorpholine.
 20. A gangliosidederivative according to claim 17, wherein the basic gangliosides areselected from the group consisting of G_(M1), G_(D1a), G_(Dlb) andG_(T1b).
 21. A ganglioside derivative according to claim 18, wherein thebasic gangliosides are selected from the group consisting of G_(M1),G_(D1a), G_(Dlb) and G_(T1b).
 22. A ganglioside derivative according toclaim 19, wherein the basic gangliosides are selected from the groupconsisting of G_(M1), G_(D1a), G_(Dlb) and G_(T1b).
 23. A gangliosidederivative according to claim 17, wherein the hydroxyl groups of saidceramide moiety and said oligosaccharide moiety are acylated with acarboxylic acid, said carboxylic acid being a substituted orunsubstituted aliphatic, aromatic, araliphatic, alicyclic orheterocyclic carboxylic acid with a maximum of 10 carbon atoms.
 24. Aganglioside derivative according to claim 18, wherein the hydroxylgroups of said ceramide moiety and said oligosaccharide moiety areacylated with a carboxylic acid, said carboxylic acid being asubstituted or unsubstituted aliphatic, aromatic, araliphatic, alicyclicor heterocyclic carboxylic acid with a maximum of 10 carbon atoms.
 25. Aganglioside derivative according to claim 19, wherein the hydroxylgroups of said ceramide moiety and said oligosaccharide moiety areacylated with a carboxylic acid, said carboxylic acid being asubstituted or unsubstituted aliphatic, aromatic, araliphatic, alicyclicor heterocyclic carboxylic acid with a maximum of 10 carbon atoms.
 26. Aganglioside derivative according to claim 20, wherein the hydroxylgroups of said ceramide moiety and said oligosaccharide moiety areacylated with a carboxylic acid, said carboxylic acid being asubstituted or unsubstituted aliphatic, aromatic, araliphatic, alicyclicor heterocyclic carboxylic acid with a maximum of 10 carbon atoms.
 27. Aganglioside derivative according to claim 17, selected from the groupconsisting of the peracetylates, perpropionylates, perbutyrrylates,maleinylates and succinylates of said esters.
 28. A gangliosidederivative according to claim 18, selected from the group consisting ofthe peracetylates, perpropionylates, perbutyrrylates, maleinylates andsuccinylates of said esters.
 29. A ganglioside derivative according toclaim 19, selected from the group consisting of the peracetylates,perpropionylates, perbutyrrylates, maleinylates and succinylates of saidesters.
 30. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and, as an active ingredient, an effectivenerve pathology treatment amount of a ganglioside derivative accordingto claim
 17. 31. A pharmaceutical composition comprising apharmaceutically acceptable carrier or diluent and, as an activeingredient, an effective nerve pathology treatment amount of aganglioside derivative according to claim
 23. 32. A method for thetreatment of pathologies of the nervous system of one affectedtherewith, comprising administering thereto an effective nerve pathologytreatment amount of a ganglioside derivative according to claim
 17. 33.A method for the treatment of pathologies of the nervous system of oneaffected therewith, comprising administering thereto an effective nervepathology treatment amount of a ganglioside derivative according toclaim
 23. 34. A ganglioside derivative comprised of an oligosaccharidemoiety formed by 1 to 5 monosaccharides, at least one sialic acid moietyand at least one ceramide moiety, the hydroxyl groups in saidoligosaccharide, sialic acid and ceramide moieties being acylated with amember selected from the group consisting of an aliphatic acid with amaximum of 6 carbon atoms, capric acid, an aromatic acid with a maximumof 6 carbon atoms, an araliphatic acid with a maximum of 6 carbon atoms,an alicyclic acid with a maximum of 6 carbon atoms, a heterocyclic acidwith a maximum of 6 carbon atoms, a hydroxy acid with a maximum of 10carbon atoms, an amino acid with a maximum of 10 carbon atoms, a dibasicacid with a maximum of 10 carbon atoms and benzoic acid substituted withmethyl, hydroxyl, amino or carboxyl.
 35. A ganglioside derivativeaccording to claim 34, wherein said acid is formic acid, acetic acid,propionic acid, butyric acid, valerianic acid, caproic acid, capricacid, benzoic acid, lactic acid, glycine, succinic acid, malonic acid,maleic acid, p-amino benzoic acid, salicylic acid, or phthalic acid. 36.A ganglioside derivative according to claim 34, wherein the basicgangliosides are selected from the group consisting of G_(M1), G_(D1a),G_(Dlb) and G_(T1b).
 37. A ganglioside derivative according to claim 35,wherein the basic gangliosides are selected from the group consisting ofG_(M1), G_(D1a), G_(Dlb) and G_(T1b).
 38. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or diluent and, as anactive ingredient, an effective nerve pathology treatment amount of aganglioside derivative according to claim
 34. 39. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand, as an active ingredient, an effective nerve pathology treatmentamount of a ganglioside derivative according to claim
 35. 40. A methodfor the treatment of pathologies of the nervous system of one affectedtherewith, comprising administering thereto an effective nerve pathologytreatment amount of a ganglioside derivative according to claim
 34. 41.A method for the treatment of pathologies of the nervous system of oneaffected therewith, comprising administering thereto an effective nervepathology treatment amount of a ganglioside derivative according toclaim 35.